The role of DT-diaphorase in bioreductive activation of mitomycin C was examined using HT-29 and BE human carcinoma cells which have high and low levels of DT-diaphorase activity, respectively. HT-29 cells were more sensitive to mitomycin C-induced cytotoxicity than the DT-diaphorase-deficient BE cell line. Mitomycin C induced DNA interstrand cross-linking in HT-29 cells but not in BE cells. Both mitomycin C-induced cytotoxicity and induction of DNA interstrand cross-links could be inhibited by pretreatment of HT-29 cells with dicoumarol. Metabolism of mitomycin C by HT-29 cell cytosol was pH dependent and increased as the pH was lowered to 5.8, the lowest pH tested. Metabolism of mitomycin C by HT-29 cytosol was inhibited by prior boiling of cytosol or by the inclusion of dicoumarol. Little metabolism was detected in BE cytosols. When purified rat hepatic DT-diaphorase was used, metabolism of mitomycin C increased as the pH was decreased and could be detected at pH 5.8, 6.4, 7.0, 7.4, but not at 7.8. Metabolism of mitomycin C was NADH dependent and inhibited by dicoumarol or by prior boiling of enzyme. An approximate 1:1 stoichiometry between NADH and mitomycin C removal was demonstrated and no oxygen consumption could be detected. Metabolism of mitomycin C by purified HT-29 DT-diaphorase was also dicoumarol inhibitable and pH dependent. The major metabolite formed during metabolism of mitomycin C by HT-29 cytosol, purified HT-29, and rat hepatic DT-diaphorase was characterized as 2,7-diaminomitosene. These data suggest that two-electron reduction of mitomycin C by DT-diaphorase may be an important determinant of mitomycin C-induced genotoxicity and cytotoxicity.