The quantities of outer and inner"halves" produced by freeze-fracturing human erythrocyte membranes have been measured by visible and fluorescence spectroscopy. Assays have been developed that are based on the use of two membrane surface markers: hemoglobin (Hb), a native marker for the cytoplasmic side of the membrane, and fluoresceinated concanavalin A (FITC-Con-A), a marker for the extracellular side. Hb absorbance is proportional to the fraction of cytoplasmic "half" membranes, and FITC fluorescence is proportional to the fraction of extracellular "halves." A procedure is described for the preparation of surface-labeled, intact erythrocytes suitable for the formation of homogeneous, planar cell monolayers of square-centimeter dimensions on polylysine-treated glass (PL-glass). Cell monolayers were frozen and fractured, and the fractions of absorbance and fluorescence in each of the two split portions determined. The PL-glass portion of membrane contained a substantially higher ratio of fluorescence to absorbance than unsplit controls, and its paired portion, a complementary lower ratio, demonstrating that the PL-glass portion was significantly enriched in extracellular "half" membrane. Experiments investigating split membrane recovery show that the double labeled membrane splitting technique is well suited to analysis of the transmembrane distribution of membrane lipids and polypeptides using methods that do not require quantitation by electron microscopy.

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