The interaction of the effect of dietary methionine on the availability of hepatic glutathione (GSH) and the effect of chronic ethanol (EtOH) consumption on the activity of the hepatic oxidation system was studied in relation to acetaminophen (ACAP) metabolism in mice. Adult male Swiss-Webster mice were pair-fed for 4 wk an EtOH-containing liquid diet that provided 50 or 100% of the methionine requirement in a 2 x 2 factorial design. Hepatic microsomal protein, relative liver weight and microsomal aniline hydroxylase activity were higher in EtOH-fed groups than in non-EtOH-fed groups. After an ACAP dose of 300 mg/kg body wt i.p., serum inorganic sulfate, endogenous hepatic methionine and GSH concentrations were lower, whereas uridine diphosphoglucuronosyltransferase activity was not changed compared to controls. GSH levels were lowered to a greater extent in the methionine-deficient groups than in methionine-sufficient groups. Incorporation of [35S]methionine into hepatic proteins was lower in all treatment groups after ACAP administration than in controls. The distribution of ACAP into the urinary sulfate conjugates was lower in methionine-deficient than in methionine-sufficient groups, and the percentage of sulfate and mercapturic acid conjugates formed as determined by high-performance liquid chromatographic analysis was higher in mice fed EtOH than in controls. Methionine deficiency compromises the normal pathways of ACAP disposition in the mouse, and chronic EtOH ingestion may potentiate this effect by increasing the amount of activated ACAP formed.