The normal asymmetric distribution of phospholipids across the plasma membrane of erythrocytes can be abolished by lysing and resealing cells in the presence of Ca2+. In the present study, using flow cytometric analysis of the binding of merocyanine 540 to monitor transbilayer phospholipid distribution, Ca(2+)-induced loss of asymmetry is shown to be independent from the aminophospholipid translocase which catalyzes movement of normally internal phospholipids from the outer to the inner leaflet of the membrane. Loss of asymmetry is rapid, temperature-sensitive, and occurs in an uninterrupted, intact bilayer, rather than by diffusion of lipids through the hemolytic pore. Addition of ATP during lysis reverses loss of asymmetry, and this restoration can be blocked by inhibitors of the aminophospholipid translocase. These results suggest that the ATP-dependent translocase is essential for recovery of asymmetry, in turn suggesting that separate mechanisms mediate the loss and the recovery of lipid asymmetry in erythrocytes.