To investigate the modifying role of the intestinal microflora in the metabolism of 1-nitropyrene (1-NP) via enterohepatic circulation, we collected bile from male Wistar rats administered [3H]1-NP orally. The bile was mixed with the intestinal contents (IC) prepared from untreated rats and the mixture was incubated anaerobically under an atmosphere of nitrogen at 37 C. Samples of the reaction mixture were removed at intervals to assay their mutagenic potential, to determine the radioactivity bound to the IC, and for analysis of the biliary metabolites. The binding of the radioactivity to the IC increased linearly as a function of time during the 1-hr incubation. The time-dependent binding does not occur with heat-treated IC and the binding was inhibited by addition of D-saccharic acid 1,4-lacton, a beta-glucuronidase inhibitor. The mutagenicity (for Salmonella typhimurium strain TA98 without S9 mix) of the bile increased early in the incubation period and then decreased very rapidly. The mutagenicity of the bile was also enhanced by treatment with a sonicated IC extract or beta-glucuronidase, but not with a heat-treated IC or aryl-sulfatase. The metabolites produced after the bile was incubated for short periods with the IC were mainly nitrohydroxypyrenes; at later times nitroreduction occurred. The level of acetylaminohydroxypyrenes, which were formed by deconjugation, did not change during the incubation. To determine the degree of contribution of the IC to the total acetylating capacity, we measured acetyltransferase activity of the IC and various organs in Wistar rats. The liver had the highest N-acetyltransferase activity among the seventeen organs examined. Considerable activity was also detected in the kidney, small intestine, lung, and testis, but the IC showed very low activity. The acetylating capacity of the IC was 0.27% of the total capacity in rats, and that of the liver was more than 80%. These results suggest that the nitrohydroxypyrenes formed from 1-NP in the liver were conjugated to glucuronic acid and excreted via the bile duct into intestine. Hydrolysis of these glucuronide conjugates by bacterial beta-glucuronidase liberated into intestine, free nitrohydroxypyrenes, which were direct-acting mutagens. The released aglycons were then rapidly nitro-reduced by intestinal microflora, but contribution of the intestinal microflora to acetylation of the reduced metabolites is very low.