E09 [3-hydroxymethyl-5-aziridinyl-l-methyl-2-(H-indole-4, 7-indione)-propenol] is a novel indoloquinone structurally related to mitomycin C, a quinone anticancer drug that requires reductive bioactivation. NAD(P)H: (quinone-acceptor) oxidoreductase (quinone reductase, DT-diaphorase, EC 1.6.99.2) is an obligate 2-electron donating enzyme that can reduce a variety of quinones resulting either in bioactivation or bioprotection. Using quinone reductase (QR) preparations from rat Walker 256 mammary tumor cells and human HT29 colon carcinoma cells, we have characterized the role of this enzyme in E09 reductive metabolism. QR activity was assayed under optimal conditions by following cytochrome c reduction at 550 nm in the presence of enzyme, quinone substrate, NADH, and bovine albumin, and confirmed by loss of E09 absorbance at 550nm. Both the rat and human tumor cell enzymes catalyzed reduction of the benchmark quinone menadione with a similar Km of 1.4-3.1 μM, although the Vmax was 7 to 8-fold lower for the human preparation. E09 was readily reduced by the rat Walker QR. The mean Km was about 5-fold higher than for menadione at around 15 μM and the Vmax was 6-fold lower at around 2.5 μmol of cytochrome c reduced mg-1 of protein. E09 was also metabolized by QR from HT29 human colon carcinoma cells but rather less efficiently than by the rat tumor enzyme. For example, the rate was 6-fold lower than that for the Walker tumor enzyme at 100 μM substrate concentration after correcting for the 7- to 8-fold difference in specific activity for the two preparations. Reduction of E09 by both the rat and the human tumor preparations was inhibited by the QR inhibitor dicoumarol (100 μM). Using highly purified rat Walker tumor enzyme, E09 was shown to be reduced to a species causing single strand breaks in pBR 322 plasmid DNA in vitro, as detected by agarose gel mobility. This activity was maximal at between 10–50 μM E09. It was inhibited completely by 10 μM dicoumarol and unaffected by superoxide dismutase up to 2000 Units mL−1. These studies show that rat and human QR reduced E09 in vitro and that for the rat enzyme this represents a bioactivation pathway which causes DNA single strand breaks.