This work investigated the oxidative injury to human red blood cells (RBCs) by the exposure to exogenous malondialdehyde (MDA), in a physiological environment. When a 10% RBC suspension was incubated in autologous plasma, in the presence of 50 microM MDA, 30% of MDA entered into the cells. A time-course study showed that MDA caused early (30-120 min) and delayed (3-18 h) effects. MDA caused a fast depletion of reduced glutathione, and loss of the glucose-6-phosphate dehydrogenase activity, followed by a decrease of HbO2. Accumulation of methemoglobin, and formation of small amounts of hemichrome were later evident. Also, an HbO2-derived fluorescent product was measured in the membrane. The redox unbalance was followed by structural and functional damage to the membrane, evident as the formation of conjugated diene lipid hydroperoxides, concurrent with a sharp accumulation of MDA, consumption of membrane vitamin E, and egress of K+ ions. SDS--PAGE of membrane proteins showed formation of high molecular weight aggregates. In spite of the marked oxidative alterations, the incubation plasma prevented a substantial hemolysis, even after a 18 h incubation. On the contrary, the exposure of RBCs to 50 microM MDA in glucose-containing phosphate saline buffer, resulted in a 16% hemolysis within 6 h. These results indicate that the exposure to MDA causes a rapid intracellular oxidative stress and potentiates oxidative cascades on RBCs, resulting in their dysfunction.