Mitomycin C (MMC) is a bioreductive antitumor agent that is activated by NADPH:cytochrome P450 reductase (EC 1.6.2.4) and NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) (DT-diaphorase). DT-diaphorase is a two-electron reducing enzyme that is induced by a variety of chemicals, including quinones. Doxorubicin (DOX) is an anthraquinone antitumor agent that has been used clinically with MMC for combination chemotherapy in breast cancer. In this study, we investigated whether DOX could selectively induce DT-diaphorase in tumor cells and whether combining this agent with MMC in an appropriate schedule could produce synergistic antitumor activity. Treatment of EMT6 murine mammary tumor cells with DOX resulted in a 40% increase in DT-diaphorase activity in these cells, but had no effect on this enzyme in murine bone marrow cells. Combination therapy with DOX and MMC produced a 1.4-fold level of synergistic cell kill in the tumor cells, but a similar level of synergy was also observed in normal bone marrow cells. Thus, DOX can selectively induce elevated levels of DT-diaphorase in tumor cells; however, the synergy observed by combining this agent with MMC appears to be unrelated to the induction of DT-diaphorase.