The aerobic and hypoxic metabolism of 2-nitroanthracene (2-NA) and 9-nitroanthracene (9-NA), two components of diesel exhaust, was studied and the mutagenicities of the parent compounds and their metabolites were compared. 2-NA was metabolized by 3-methylcholanthrene-induced rat liver microsomes under aerobic conditions to 2-NA trans-5,6-dihydrodiol, 2-NA trans-7,8-dihydrodiol, 2-NA 7-keto-5,6,7,8-tetrahydro-trans-5,6-diol, 2-NA 6-keto-5,6,7,8-tetrahydro-trans-7,8-diol, 2-nitro-9,10-anthraquinone and 2-NA 5,6,7,8-tetrahydrotetrol. When incubations were conducted under hypoxic conditions, 2-aminoanthracene was produced facily. N.m.r. spectral analysis indicated that the nitro-substituent of 2-NA and all of its ring-oxidized metabolites preferentially adopted an orientation in which the nitro group was coplanar or nearly co-planar with the aromatic ring system. 2-NA and its two trans-dihydrodiol metabolites were mutagenic in Salmonella typhimurium strain TA98, both in the presence and in the absence of S9 enzymes while the two tetrahydrodiol-ketones were much less mutagenic. When assayed in strains TA98NR and TA98/1,8-DNP6, the mutagenic activities of 2-NA and the trans-7,8-dihydrodiol were decreased. 2-Aminoanthracene was mutagenic in strain TA98 only in the presence of S9 enzymes. When 2-aminoanthracene was metabolized aerobically, the corresponding trans-5,6- and 7,8-dihydrodiols were not detected. These results suggest that 2-NA can be metabolized to mutagenic products by nitroreduction and ring-oxidation followed by nitroreduction, but not nitroreduction followed by ring-oxidation. Aerobic metabolism of 9-NA produced 9-NA trans-1,2- and 3,4-dihydrodiols, while metabolism was not detected under anaerobic conditions. Previous studies indicated that 9-NA and its two metabolites were not mutagenic in TA98. The differences in the orientation of the nitro substituents in 2-NA and its ring-oxidized metabolites and in 9-NA and its metabolites can be employed to explain the strong mutagenicity of 2-NA and weak mutagenicity of 9-NA when assayed both in the absence and in the presence of S9 activation enzymes.