The metabolism of [14C]-1-nitropyrene by human, rat and mouse intestinal microflora and a bioassay-directed chemical analysis of the isolated metabolites by assaying HPLC fractions with a microsuspension reverse mutation assay were examined. [14C]-1-Nitropyrene was metabolized by human, rat, and mouse intestinal microflora to 1-aminopyrene, N-acetyl-1-aminopyrene, N-formyl-1-aminopyrene, and two unknown metabolites identified as A and B. The predominant metabolite produced by human, rat, or mouse intestinal microflora following a 12-h incubation with [14C]-1-nitropyrene was 1-aminopyrene, which accounted for 93, 79, and 88% of the total 14C, respectively. Only minor amounts of N-formyl-1-aminopyrene (1.4, 1.2, and 1.0%), N-acetyl-1-aminopyrene (4.4, 3.0, and 3.4%), unknown A (1.0, 1.2, and 1.0%), and unknown B (3.3, 5.0, and 1.2%) were detected. These data suggest that a similar mechanism exists in the biotransformation of 1-nitropyrene by intestinal microflora from all three sources. Direct mutagenicity analysis of the HPLC fractions produced by intestinal microflora with the microsuspension reverse mutation assay indicated that mutagenic fractions can be resolved using this methodology.