Among the nitro-substituted polycyclic aromatic hydrocarbons identified in environmental samples and known to be genotoxic, 1-nitropyrene is one of the most abundant. The biotransformation of 1-nitro((14)C)pyrene by human, rat, and mouse intestinal microflora and the mutagenicity of the isolated metabolites by direct analysis of the HPLC fractions with a microsuspension mutation assay were investigated. 1-nitro((14)C)pyrene was metabolized by human, rat and mouse intestinal microflora to the following reductive metabolites; 1-aminopyrene, Nacetylaminopyrene, N-formyl-1-aminopyrene and two unknown metabolites identified as A and B. The predominant metabolite of 1-nitro((14)C)pyrene produced by human, rat or mouse intestinal microflora following a 12 h incubation was 1-aminopyrene which accounted for 79 to 93% of the total (14)C respectively. Only minor amounts of N-formyl-1-aminopyrene (1%), N-acetylaminopyrene (3 - 4%) were produced. The similarity in the distribution of the reductive metabolites suggests that a similar mechanism exists in the biotransformation of 1-nitropyrene by intestinal microflora of different mammalian species.