To study the effect of chronic ethanol consumption on biliary excretion of acetaminophen, male Sprague-Dawley rats were pair-fed a nutritionally adequate liquid diet containing either ethanol or isocaloric carbohydrate for 4-6 weeks. One day after bile duct cannulation, acetaminophen (250 mg/kg body weight) was given ip and drug metabolism was studied for 5 hr. Biliary excretion of acetaminophen and its principal metabolites was significantly decreased in rats fed alcohol chronically when compared to pair-fed controls. Although no difference was found in the urinary excretion of the drug, the total elimination (biliary + urinary) was significantly depressed in the alcohol-fed animals. The decreases in hepatic glutathione content and in biliary glutathione concentration (after acetaminophen injection) were significantly greater in ethanol-fed rats than in controls. These data support the possibility that diminished hepatic glutathione and biliary excretion of the drug may contribute to the increased hepatotoxicity of acetaminophen after chronic alcohol feeding.