Nitro-substituted polycyclic aromatic hydrocarbons (N02PAH) such as 1-nitropyrene (NP) and 3-nitrofluoranthene (3-NFA), both widespread environmental mutagens, generally require activation by bacterial nitroreductases for maximal expression of their mutagenicity in the Ames Salmonella histidine reversion assay; addition of exogenous mammalian drug-metabolishing enzymes (S9 fraction) decreases their activity in the system. In contrast, in an assay of forward mutation (to 8-aza-guanine resistance) with Salmonella typhimurium strain TM677, without S9 NFA has relatively low activity (10 to 20 mutants per 10 to the 5th power survivors at 5 micrograms/ml) and NP only marginally doubles the background mutation rate. Addition of S9 protein enhances NP at 50 micrograms/ml and 80 to 100 x 10 to the minus 5 power for NFA at 5 to 10 micrograms/ml. Therefore generation of active genotoxic intermediates in the forward mutation system may proceed through metabolic pathways other than the previously-defined bacterial nitro-reduction. Previously-identified oxidative metabolites of NP are known to be mutagenic.