3-Nitrobenzanthrone (3-NBA) is a potent mutagen and potential human carcinogen identified in diesel exhaust and ambient air particulate matter. Previously, we detected the formation of 3-NBA-derived DNA adducts in rodent tissues by 32P-postlabeling, all of which are derived from reductive metabolites of 3-NBA bound to purine bases, but structural identification of these adducts has not yet been reported. We have now prepared 3-NBA-derived DNA adduct standards for 32P-postlabeling by reactingN-acetoxy-3-aminobenzanthrone (N-Aco-ABA) with purine nucleotides. Three deoxyguanosine (dG) adducts have been characterised asN-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-C8-N-ABA), 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-N2-ABA) and 2-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone-3′-phosphate (dG3′p-C8-C2-ABA), and a deoxyadenosine (dA) adduct was characterised as 2-(2′-deoxyadenosin-N6-yl)-3-aminobenzanthrone-3′-phosphate (dA3′p-N6-ABA). 3-NBA-derived DNA adducts formed experimentallyin vivoandin vitrowere compared with the chemically synthesised adducts. The major 3-NBA-derived DNA adduct formed in rat lung cochromatographed with dG3′p-N2-ABA in two independent systems (thin layer and high-performance liquid chromatography). This is also the major adduct formed in tissue of rats or mice treated with 3-aminobenzanthrone (3-ABA), the major human metabolite of 3-NBA. Similarly, dG3′p-C8-N-ABA and dA3′p-N6-ABA cochromatographed with two other adducts formed in various organs of rats or mice treated either with 3-NBA or 3-ABA, whereas dG3′p-C8-C2-ABA did not cochromatograph with any of the adducts foundin vivo. Utilizing different enzymatic systemsin vitro, including human hepatic microsomes and cytosols, and purified and recombinant enzymes, we found that a variety of enzymes [NAD(P)H:quinone oxidoreductase, xanthine oxidase, NADPH:cytochrome P450 oxidoreductase, cytochrome P450s 1A1 and 1A2,N,O-acetyltransferases 1 and 2, sulfotransferases 1A1 and 1A2, and myeloperoxidase] are able to catalyse the formation of 2-(2′-deoxyguanosin-N2-yl)-3-aminobenzanthrone,N-(2′-deoxyguanosin-8-yl)-3-aminobenzanthrone and 2-(2′-deoxyadenosin-N6-yl)-3-aminobenzanthrone in DNA, after incubation with 3-NBA and/or 3-ABA.