Retinal pigment epithelial cell plasma membranes were isolated from the eyes of normal and RCS-dystrophic rats by binding glass microbeads to the intact pigment epithelial cell layer, removal of the bead-bound cells from the eyes and subsequent sucrose density gradient centrifugation. Plasma membranes were recovered from the gradients in identical yields and characterized by membrane marker enzymes, lipid analysis and SDS-polyacrylamide gel electrophoresis. Membrane purification by alkaline phosphodiesterase I and 5'nucleotidase activities averaged 8-fold for normal rats and 5.5 for the dystrophic rats. The ratio of cholesterol per microgram protein indicated 6 to 7-fold purification for both types of plasma membranes. Na+K+-ATPase in the normal and mutant rat plasma membranes was purified 5- and 3.5-fold, respectively, but the specific activities of both Na+K+-ATPase and 5'nucleotidase were higher in the dystrophic rat membranes than in normal. Subcellular organelle contamination was low and relatively uniform in both types of membranes, while opsin contamination was less than 1%. By electrophoretic analysis the plasma membrane proteins were similar, with 30-40 identifiable bands present in each membrane type. The plasma membranes both contain high levels of cholesterol, sphingomyelin and phosphatidylcholine and low levels of polyunsaturated fatty acids. However, the dystrophic rat membranes had significantly higher levels of docosahexaenoic acid than normal, and significantly lower levels of arachidonic acid. The differences in these plasma membrane fatty acids and in the membrane-bound enzymes may affect the ionic balance of the interphotoreceptor matrix or otherwise contribute to degenerative changes in dystrophic rat photoreceptors.