The incidence of hepatocellular carcinoma is higher in male rats fed 35 mg of dinitrotoluene (DNT) per kg than in female rats fed the same dose. Sex differences in DNT disposition and/or metabolism may account for this difference. This study characterized the metabolism and biliary excretion of 2,4-DNT in male and female isolated perfused rat livers. Livers from both sexes displayed a capacity for oxidation, reduction, acetylation and conjugation of 2,4-DNT (or its metabolites). Oxidation of 2,4-DNT to 2,4-dinitrobenzyl alcohol followed by glucuronidation to 2,4-dinitrobenzyl alcohol glucuronide (DNBalcG) was the major route of 2,4-DNT metabolism in both sexes. Formation of DNBalcG accounted for 10 to 30% of the total initial 2,4-DNT concentration in perfusates. After perfusion with 20 microM 2,4-DNT, male livers excreted larger quantities of DNBalcG in the bile (392 nmol) than female livers (172 nmol); at the same 2,4-DNT concentration, perfusates from female livers contained over 3 times as much DNBalcG as male perfusates. These data suggested that female livers transported DNBalcG into the bile at slower rates than male livers. The transport of DNBalcG into the bile of male, but not female, livers appeared to be saturated after perfusion with 20 micro M 2,4-DNT. No sex differences in the hepatic macromolecular covalent binding were observed after perfusion of livers with either 20 or 70 micro M 2,4-DNT. These data suggest that the major difference in the in vitro metabolism of 2,4-DNT between male and female rats is the larger quantities of DNBalcG excreted in the bile of male rats than female rats.