The osmotic behavior of rat liver mitochondria has been studied in a sucrose medium. The mitochondria behave like a two compartment system. One compartment is permeable to sucrose and has a volume of 1.22 µl/(mg mitochondrial dry weight) in a 272 milliosmol sucrose medium; the second, inaccessible to sucrose, has a volume of 0.555 µl/mg dry weight) under the same conditions. Part of the water in the sucrose inaccessible space is apparently not free to participate in osmotic phenomena. This volume is 0.272 µl/(mg dry weight) under the same conditions. It is suggested that the osmotically inactive water corresponds to the water of hydration of the mitochondrial macromolecules. The volume of the remainder of the water in the sucrose inaccessible space depends inversely on the osmolality of the medium, as is to be expected. The volume of water in the sucrose accessible space is constant, independent of the osmolality of the medium, as is the volume of the mitochondrial framework plus the nonvolatile solutes.

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