1- and 3-nitrobenzo[a]pyrene (1- and 3-nitro-BaP) are environmental pollutants and are S9-mediated mutagens in the Chinese hamster ovary (CHO) cell/hypoxanthine-guanine phosphoribosyl transferase assay. In this study, we examined the pathways leading to the mutagenic activation of these compounds in CHO cells. The microsomal metabolites of 1- and 3-nitro-BaP, the 1- and 3-nitro-BaP trans-7,8-dihydroxy-7,8-dihydrodiols (trans-7,8-dihydrodiols) and the 1- and 3-nitro-BaP trans-9,10-dihydrodiols, were isolated and tested for mutagenicity. At the concentrations assayed, both trans-9,10-dihydrodiols were non-mutagenic with and without S9 activation. In contrast, the trans-7,8-dihydrodiols of 1- and 3-nitro-BaP were direct-acting mutagens and these responses were similar in magnitude to the S9-mediated mutagenicities of the parent nitro-BaPs. S9 increased the mutagenic responses of the trans-7,8-dihydrodiols approximately 20-fold. Inhibition of epoxide hydrolase decreased the S9-mediated mutagenicity of 1-nitro-BaP by half, but doubled the S9-mediated mutagenicity of 3-nitro-BaP. These results suggest that in CHO cells: (i) the major route of mutagenic activation of 1- and 3-nitro-BaP involves S9-generated derivatives of the trans-7,8-dihydrodiols, e.g. bay-region diol epoxides; (ii) reactive nitroarene oxides may contribute to mutation induction by 3-nitro-BaP; and (iii) metabolic routes involving trans-9,10-dihydrodiol formation result in detoxification.