A survey has been made of the amount of muscle water available to inulin, sucrose, and radioiodinated human serum albumin (RISA). The percentage spaces available to the three molecules are of the same order of magnitude, but the sucrose space > inulin space > albumin space. The kinetics of influx and efflux of RISA have been studied, and it appears that a small part of the albumin may be adsorbed in the extracellular phase. Nevertheless the albumin space would appear to give the best index of the extracellular volume. The scatter in values found for the extracellular space by all methods is very great, ranging from 8 to 40 per cent and renders invalid the use of a mean value for the calculation of intracellular concentrations. The variation within paired muscles is less than between pairs, provided the tissue has undergone no volume change. Increase in total muscle volume when the muscle is placed in a hypotonic solution leads to a decrease in the size of the extracellular space.

The partition of Li + , Br - , and I - across the membrane of the sartorius muscle of the toad Bufo marinus has been investigated both at the steady state and with kinetic methods. Li + was found to have access to an amount of muscle water similar to that of Na + . Br - and I - could be regarded as being interchangeable with cellular Cl - . None of the foreign ions caused significant losses of cellular K + . Li + efflux from the cell was slower in muscles which were equilibrated for long periods in Li + than in short equilibrated muscles. Na + efflux from Li + -treated muscles was similar in rate to normal controls, but the amount of Na + in the slow fraction was increased by Li + . I - efflux was extremely rapid, and it was not possible to differentiate kinetically between intra- and extracellular material. These results have been found to be consistent with the hypothesis of a three phase system for muscle.

The partition of sulfate, Ca ++ , and Mg ++ across the membrane of the sartorius muscle has been studied, and the effect of various concentrations of these ions in the Ringer solution on the cellular level of Na + , K + , and Cl - has been determined. The level of the three divalent ions in toad plasma and muscle in vivo has been assayed. Muscle was found to contain an almost undetectable amount of inorganic sulfate. Increases in the external level of these ions brought about increases in intracellular content, calculated from the found extracellular space as determined with radioiodinated serum albumin or inulin. Less of the cell water is available to sulfate than to Cl - , and the Mg ++ space is less than the Na + space. An amount of muscle water similar to that found for Li + and I - appears to be available to these divalent ions. Sulfate efflux from the cell was extremely rapid, and it was not found possible to differentiate kinetically between intra- and extracellular material. These results are consistent with the theory of a three phase system, assuming the muscle to consist of an extracellular phase and two intracellular phases. Mg ++ and Ca ++ are adsorbed onto the ordered phase, and increments in cellular content found on raising the external level are assumed to occur in the free intracellular phase.

The Na + , K + , and inorganic phosphate levels of the plasma and sartorius muscle of the toad Bufo marinus were determined. Soaking in normal Ringer brought about the usual cation shifts, but did not alter the level of inorganic phosphate in the cell. Increases in the external phosphate level brought about an increase in the internal phosphate, but the apparent phosphate space of muscle is somewhat smaller than the apparent Cl - space. Phosphate spaces were compared with inulin spaces and were found to be significantly greater. Alteration of the H + concentration of the high phosphate Ringer did not alter the partition of phosphate across the cell membrane. These results have been found to be consistent with the theory of a three compartment system for muscle, wherein the tissue is assumed to consist of an extracellular phase, and two intracellular phases. The inorganic phosphate of the cell is assumed to be adsorbed onto the "ordered phase," and increments in organic phosphate found on raising the external level are assumed to take place in the "free intracellular phase."

The Na, + Cl - , and K + content of toad plasma and the sartorius muscle has been determined. Although the Na + and Cl - level of the muscles in the living animal varied greatly (0 to 38.0 m.eq. per kg., and 0 to 31.8 m.eq. per kg. respectively) the K + level was subject to a smaller variation (76.5 to 136 m.eq. per kg.). There was a direct relationship between Na + and Cl - , which was independent of the K + level. There is a closely related gain of Na + and Cl - when muscle is soaked in normal Ringer. These gains are not related to the K + loss, frequently found on soaking. The relationship between the three ions was studied in a large series of 124 muscles in normal Ringer. As found in vivo , there was a correlation between Na + and Cl. - This correlation was independent of K + content, except when this was abnormally low. Alteration of the external NaCl level produced concomitant changes in the internal levels of these ions. Alteration of the external KCl level produced an increase in internal Cl - similar to that found with high NaCl solutions, but the amount of K + entering the cell was approximately one-third of the external increase. Removal of K + from the external solution did not result in a loss of K + from the cell, although there was an adequate amount of Cl - present to accompany it. The results cannot be reconciled with either a Donnan concept for the accumulation of K + , or a linked carrier system. A theory is proposed to account for the ionic differentiation within the cell. The K + is assumed to be adsorbed onto an ordered intracellular phase. The normal metabolic functioning of the cell is necessary to maintain the specificity of the adsorption sites. There is another intracellular phase, which lacks the structural specificity for K + , and which contains Na + , Cl - , and K + in equilibrium with the external solution. The dimensions of the free intracellular phase will vary from cell to cell, but it will be smaller in the intact animal, and will increase on soaking in normal Ringer, until it is approximately one-third of the total cellular volume. The increase in this phase may be ascribed to a decrease in the energy available to maintain the ordered phase.

The resting and action potentials of sartorius muscles of the toad, Bufo marinus , have been measured under varying conditions of external environment. At the same time, analyses for Na + and K + content were carried out. There was a slight elevation of 2 mv. when the measurements were made in phosphate-Ringer instead of in bicarbonate-Ringer. The R.P. was independent of the hydrogen ion concentration between pH 6.5 and 8.5, although at these pH's there was marked alteration in the level of Na + and K + in the muscle. Alteration of the external K + level between 0 and 50 m.eq./liter has little influence on the internal K + concentration. When the log of the external K + concentration is plotted against the R.P. there is not a linear relationship until the external K + is raised above 12 m.eq./liter, at which point the cell is unexcitable. Above this value a straight line with a slope of 58 mv. per ten-fold change in concentration is obtained, but the absolute values at any point are about 35 per cent higher than those which would be given by the Nernst equation. Alteration of the external Na + level within a range of 45 to 650 m.eq./liter resulted in marked changes in the internal Na + content, without, however, having any effect on the ratio Na + out /Na + in . This ratio has remained at about 3 in spite of marked fluctuations in the absolute value of the internal and external Na + levels. When the Na + level is lowered there is a decrease in the height of the action potential although there is no alteration in the ratio Na + out /Na + in . As the Na + level is raised the height of the action potential is not affected even in the presence of a fivefold increase in Na + in the Ringer. The results do not support the conclusion that the bioelectric potentials can be calculated from the ionic ratios by means of simple physical chemical hypotheses such as the Nernst or Goldman equations. The maintenance of the normal K + content of the cell cannot be accounted for by a Donnan mechanism. No definite evidence has been produced to explain the mechanism of a Na + "pump." In other words, the concept of a Na + pump requires that there shall be a physico- or organochemical mechanism which will distinguish between Na + and K + (or other) ions. There is evidence that Na + can be extruded against a concentration gradient. On the other hand the cell is able to maintain a constant ratio of external to internal Na + even when the cell has been severely damaged by very high external Na + levels.

The sartorius muscles of 320 toads have been analyzed for Na + and K + . There is a wide variation in the Na + content which when calculated intracellularly varied from 0 m.eq./kg. to 58 m.eq./kg. In particular it was found that the distribution of internal Na + in the intact animal was such that only 17 per cent of the muscles should give from the Nernst equation the observed overshoot of 37 mv. In contrast to this wide variation the K + content is comparatively constant, the range being 71 to 112 m.eq./kg. The mean observed resting potential of 87 mv. agreed well with the potential calculated from the mean intracellular K + by the Nernst equation. Analyses of plasma show that the Na + content is constant at 130 m.eq./liter, and the K + is 3.0 m.eq./liter. The resting and action potentials of 77 muscles have been recorded and then the muscles have been analyzed. The results have shown that there is no correlation between the level of intracellular Na + and the overshoot. Furthermore the apparent correlation between the average K + content and the average resting potential has been shown to be fortuitous, when the correlation in individual muscles is considered. When a muscle is soaked in Ringer solution for several hours there is a gain of Na + and a loss of K + . These shifts should result in changes in the respective potentials, but such changes were not found. The above findings have been discussed in the light of the present theories that the resting potential and the action potential are directly related to the ionic ratio across the membrane. Our results very definitely do not support the theory that the overshoot is related to the Na + gradient, and this also applies with respect to the K + gradient and the resting potential.