Human erythrocytes were separated into three density groups representing different age groups. Phospholipid outside-inside translocation rates and equilibrium distribution were determined in each group with spin-labeled phosphatidylserine (PS*), phosphatidylethanolamine (PE*), and phosphatidylcholine (PC*), at 37 degrees C and 4 degrees C. At both temperatures, the initial velocity of aminolipid translocation was reduced in the more dense (older) cells. The equilibrium distribution was not significantly modified for PS*, but a larger fraction of PE* remained on the outer monolayer of the more dense cells. PC* transmembrane diffusion was identical in the three fractions. Cytosolic ATP, which is required for aminophospholipid translocation, was not responsible for the variability of the density separated cells since ATP enrichment did not cancel the differences between top and bottom fractions, although it equalized the ATP concentration of the various fractions. Variations in the level of intracellular Ca2+ could also be excluded. Thus, the enzyme aminophospholipid translocase seemed to be directly altered in aged cells, possibly due to oxidation caused by lipid peroxidation products. Experiments with malonyldialdehyde or H2O2 treated cells confirmed this interpretation and suggest that defects in endogenous lipid asymmetry observed in aged human erythrocytes may be due to altered activity of the translocase.