The dietary and environmental agent, 6-nitrochrysene (6-NC) is a powerful mammary carcinogen and mutagen in rats. It is known to be metabolized by ring-oxidation, nitro-reduction and a combination of the two pathways. In order to determine the ultimate mutagenic metabolites, we have compared the previously determined mutational profile of 6-NC in rat mammary gland [T. Boyiri, et al. (2004) Carcinogenesis, 25, 637-643] with that of five of its known metabolites in the cII gene of lacI mammary epithelial cells in vitro. In vivo, 6-NC gives rise to three major mutations, AT > GC, AT > TA and GC > TA (in decreasing order) which comprise >70% of the mutations. The metabolite whose mutational profile was most similar to that of 6-NC in vivo was trans-1,2-dihydroxy-1,2-dihydro-N-hydroxy-6-aminochrysene (1,2-DHD-6-NHOH-C) which arises from both ring-oxidation and nitro-reduction. However, metabolites arising from either ring-oxidation or nitro-reduction alone exhibited some similarities to mutational profile of 6-NC. These results, taken in conjunction with previous data showing that the major DNA adducts in mammary tissue of rats treated with 6-NC are products of the reaction of 1,2-DHD-6-NHOH-C with guanine and adenine, make a strong case that 1,2-DHD-6-NHOH-C is the ultimate genotoxic metabolite from 6-NC.