In this paper we demonstrate that NADPH-initiated oxidative damage of microsomal proteins occurs in the absence of free metal ions and that this protein oxidation is mediated by cytochrome P450 (cyt P450). Oxidized proteins are rapidly degraded by proteases. Ascorbate (AH2) specifically inhibits free metal ion-independent cyt P450-mediated protein oxidation and thereby prevents subsequent proteolytic degradation. Other scavengers of reactive oxygen species including superoxide dismutase, catalase, and glutathione are ineffective. This is in variance with free metal ion-catalyzed protein oxidation, which is accelerated by AH2 and inhibited by catalase. Oxidative damage of proteins has been assessed by the production of carbonyl groups, bityrosine formation, and tryptophan loss. The mechanism of protein oxidation has been studied using a reconstituted system comprised of purified NADPH-cyt P450 reductase, cyt P450, and isolated microsomal proteins as well as model polypeptides, e.g. poly-L-proline and poly-L-lysine. Cyt P450 Fe3+ is reduced by NADPH-cyt P450 reductase to cyt P450 Fe2+, which consumes oxygen in a stoichiometric proportion to produce cyt P450 Fe2+ O2, the resonance form of which is a perferryl moiety, cyt P450 Fe3+.O2-.. It is proposed that cyt P450 Fe3+.O2-. abstracts hydrogen from amino acid side chains leading to the production of carbonyl derivatives. Tentatively, AH2 prevents protein oxidation by interacting with cyt P450 Fe3+.O2-..