1-Nitropyrene (1-NP), present in diesel engine emissions, is a potent mutagen to bacteria, such as those found in mammalian intestinal tract, which contain nitroreductase enzymes. The purposes of this study were to determine the importance of bile as a route of excretion of 1-NP metabolites and to determine if reabsorption of biliary metabolites required the presence of intestinal bacteria. The bile ducts of male Fischer-344 rats were cannulated, 0.3 or 1.2 mumoles [3H]1-NP was given i.v., and bile, urine, and feces were collected for 24 hr. Biliary excretion accounted for 70 (80%) or 170 (60%) nmoles of [3H]1-NP after the low and high dose, respectively, with half-times for excretion of 1.7 hr +/- 0.3 (+/- S.E.M.) and 3.4 hr +/- 1.6 (+/- S.E.M.). Excretion of [3H]1-NP equivalents in the urine was linearly related to dose, with 6 or 16 nmoles (8%) excreted in 24 hr. At the low dose, more radioactivity appeared in the urine in control rats compared to bile-duct cannulated rats, suggesting that reabsorption of 1-NP metabolites occurred. Pretreatment of rats with orally administered antibiotics prior to i.v. injection of 0.3 mumole [3H]1-NP decreased radioactivity excreted in urine compared to untreated controls, suggesting that intestinal microorganisms may alter the biliary metabolites of 1-NP to facilitate reabsorption. Pretreatment of rats with buthionine sulfoximine, a glutathione depletor, decreased the excretion of certain biliary metabolites, suggesting that they were mercapturic acids of 1-NP metabolites. In summary, the results of these studies indicate that bile was an important route of excretion of nitropyrene metabolites. A portion of the excreted metabolites was reabsorbed from the gut, and this reabsorption required the presence of gut microorganisms.