1-Nitropyrene (NP), an environmental pollutant, a potent mutagen and an animal carcinogen, undergoes reduction, acetylation, ring-hydroxylation and conjugation in the rat in vivo to form mutagenic metabolities which are excreted in the urine. In order to investigate the role of the gut flora in the generation of these metabolities, germ-free rats of the AGUS strain, and conventional AGUS rats matched for sex and age, where injected intraperitoneally with NP labelled with (14)C. When urines were examined for mutagenicity with the Ames plate incorporation assay, the highest mutagenic activity was seen in 8-24 h urine from conventional rats in the presence of S9. These results indicate that the gut flora are necessarity involved in the formation of 6-hydroxy-1-acetamidopyrene, and their role may include both introreduction and the hydrolysis of glucuronides released from enterohepatic recirculation.