The direct-acting mutagenicity of 5-nitroacenaphthene for Salmonella typhimurium is dependent upon the reduction of the nitro function as evidenced by the significant decrease in mutagenicity seen with nitroreductase-deficient Salmonella strains. Addition of microsomal preparations results in a significant increase in mutagenicity and a by-passing of the block in nitroreductase-deficient and arylhydroxylamine esterifying-deficient enzyme strains. The results are taken to indicate that the microsome-induced mutagenicity is due primarily to oxidation of the acenaphthene moiety. The results are consistent with recent studies which indicate that the nitro function exercises a directing effect on ring oxidation.