5-Nitroacenaphthlene induces primarily mutations of the frameshift type in Salmonella typhimurium (i.e., strains TA98 and TA1538). Addition of activation mixtures (S9) results in an increased mutagenic potency as well as a shift in specificity to base-substitution mutations (i.e., strain TA100). The magnitude of these effects depends upon the species of origin of the S9 and whether the animals were induced (Aroclor 1254). These effects are ascribed to the S9-mediated conversion of 5-nitroacenaphthene to additional mutagenic metabolites (possibly ring oxidation products). In addition, while 5-nitroacenaphthene exhibited appreciable activity in the absence of S9 for strain TA98, its mutagenicity for TA97, also an indicator of frameshifts, was negligible. TA97 differs from TA98 in the nucleotide sequence at the mutational hot spots (a run of 6 cytosine as opposed to 8 alternating guaninecytosine residues). These findings indicate that a careful analysis of the results obtained in the standard Salmonella typhimurium mutagenicity assay may yield useful information regarding bioconversion pathways and the role of DNA sequence in the ultimate expression of pro-mutational events.