Phospholipid asymmetry in biological membranes is maintained by an aminophospholipid-specific Mg(2+)-ATPase that transports PS and PE from the outer to the inner monolayer. Recent evidence indicates that a loss of phospholipid asymmetry occurs in erythrocytes from diabetic individuals, resulting in the appearance of PS in the membrane outer leaflet. We show that hyperglycemic treatment of normal erythrocytes duplicates this effect. Erythrocytes incubated for 18-24 h in the presence of glucose were assayed for PS transport and transmembrane phospholipid asymmetry. Phospholipid asymmetry in erythrocytes treated with high concentrations of glucose (> 5 mM) showed a time-dependent (t1/2 approximately 12 h) and concentration-dependent (half-maximal concentration approximately 7.5 mM) increase in the accessibility of PS and PE, and a decrease in the accessibility of SM and PC, to exogenous phospholipases. After an 18 h incubation with 20 mM glucose, 40% of the endogenous PS and PE was found in the outer monolayer concomitant with a decrease in the outer monolayer content of SM (from 80% to 50%) and PC (from 75% to 65%). These values are consistent with an almost complete transbilayer scrambling of erythrocyte phospholipids. The loss of PS asymmetry was verified using an assay based on the activation of the prothrombinase complex. The observed loss of asymmetry is not due to inhibition of PS transport or glucose-induced Ca2+ influx.(ABSTRACT TRUNCATED AT 250 WORDS)