Treatment of MCF-7 cells with doxorubicin in the presence of purified rat NADPH cytochrome P450 reductase (P450red) and NADPH resulted in a marked enhancement of drug cytotoxicity. No potentiation of cell killing was observed when the drug was incubated with P450red and NADPH prior to addition to the cells, implicating the involvement of short-lived species. The increase in doxorubicin toxicity was clearly related to its metabolism by P450red. Of a variety of free radical scavenging agents tested only glutathione was effective in protecting against the toxic metabolites produced by reductive activation. Preliminary experiments also showed that doxorubicin binding to cellular proteins and DNA occurred to a greater extent when cells were treated with the drug and P450red. Our data indicate that oxygen radicals do not play a primary role in cytochrome P450 reductase-mediated doxorubicin cytotoxicity and suggest that, in this case, the mechanism of toxicity involves a reactive species which binds to cellular nucleophiles.