Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway.