The formation and biliary excretion of the glutathione and cysteine S-conjugates of hexachlorobutadiene were studied in the isolated perfused rat liver. Infusion of increasing amounts of hexachlorobutadiene led to an increase in total metabolite excretion. Partitioning of glutathione conjugate release between bile and perfusate depended on the rate of substrate infusion: S-(1,2,3,4,4-pentachlorobutadienyl)glutathione (PCBG) appeared quantitatively in bile at low hexachlorobutadiene infusion rates, and increasing amounts of the glutathione conjugate were found in the perfusate as infusion rates were increased. The cysteine S-conjugate, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine, was not detected in the perfusate, but the amounts found in the bile were correlated with the concentrations of the glutathione conjugate. Depletion of hepatic glutathione concentrations decreased PCBG formation. Hence, at moderate hexachlorobutadiene infusion rates, PCBG is exclusively excreted into bile, indicating that intestinal absorption of PCBG or its metabolites is required for the induction of kidney damage in vivo.