Human erythrocytes were incubated with sonicated vesicles composed of diheptanoyl-, dioctanoyl-, didecanoyl-, or dimyristoylphosphatidylserine, and the transbilayer distribution of the incorporated foreign lipid was examined by monitoring changes in cell morphology (Daleke & Huestis (1989) J. Cell. Biol. 108, 1375). Cells incubated with all phosphatidylserine homologs crenated initially and then reverted to discoid and stomatocytic morphology. Cells exposed to didecanoyl- or dimyristoylphosphatidylserine retained stable stomatocytic morphology during more than 10 h of incubation at 37 degrees C. Cells exposed to the diheptanoyl or dioctanoyl homologs reverted from stomatocytes to discocytes within 1-4 h. This reversion was more rapid for the shorter acyl chain diheptanoylphosphatidylserine. Reversion was accelerated in both cases by vanadate, an inhibitor of the aminophospholipid translocator. Heat denaturation of cytoskeletal proteins had no effect on phosphatidylserine-induced stomatocytosis or on the reversion to discoid shape of cells exposed to the short-chained homologs. These observations suggest that the aminophospholipid transporter rather than cytofacial lipid binding sites plays the primary role in maintenance of phosphatidylserine asymmetry in the erythrocyte membrane bilayer.