The metabolism and excretion of the hepatocarcinogen 2,6-dinitro-toluene (2,6-DNT) was investigated in Fischer-344 rats in vivo and in isolated perfused livers. Urinary excretion accounted for half of the dose (10 mg/kg) 72 hr after administration of 14C-2,6-DNT. 2,6-Dinitrobenzoic acid, 2,6-dinitrobenzyl alcohol glucuronide (2,6-DNBAlcGluc), and 2-amino-6-nitrobenzoic acid accounted for 95% of the urinary 14C. Fecal excretion accounted for one-fifth of the dose in 72 hr. 2,6-DNBAlcGluc was the major metabolite found in the perfusate and bile of isolated perfused rat livers. Biliary excretion of 2,6-DNBAlcGluc by livers from male rats was 3.3- and 8.6-fold that of female rats on perfusion with 20 and 70 microM 14C-2,6-DNT, respectively. No sex-dependent differences in biliary flow rates were observed. Twice as much 14C was found to be covalently bound to hepatic macromolecules in male than female rat livers in vivo. Decreased biliary excretion of 2,6-DNBAlcGluc may account for the lesser amount of 14C found to be covalently bound in female rat livers. Observations with 2,6-DNT parallel those made with 2,4-DNT, suggesting that both isomers are metabolized by the liver, excreted in the bile, deconjugated and further metabolized by the intestinal microflora, and transported back to the liver for, perhaps, additional metabolism and covalent binding to an endogenous component of the liver.