Antioxidant activity of exogenous cytochrome c was investigated in vitro using the whole brain homogenate, mitochondrial fraction and postmitochondrial supernatant containing microsomes prepared from rat brains. Increments in the amount of lipid peroxides were observed in each fraction when incubated at 30 degrees C, while the addition of cytochrome c (200 mM) effectively suppressed the production of peroxides. This depressive effect of cytochrome c was more prominent in the supernatant than in the mitochondrial fraction. Although the peroxidation was enhanced markedly by the addition of NADPH (2 mM), particularly in the mitochondrial fraction, cytochrome c was able to prevent its acceleration. This inhibitory mechanism might be explained by the fact that cytochrome c deprived superoxide radicals of electrons generated in ischemic insult. The results of the present study suggest that exogenous cytochrome c has free radical scavenging or antioxidant activity, which might be responsible in part for its cerebral protective action during ischemia.