The mechanism of ethanol-induced depletion of hepatic glutathione (GSH) was studied in vivo and in isolated hepatocytes. Neither inhibition of ethanol metabolism with 4-methylpyrazole, nor a 10-fold elevation of acetaldehyde levels by inhibition of aldehyde dehydrogenase with cyanamide or disulfiram, affected the magnitude of the GSH depletion observed in vivo. The rate of intracellular GSH accumulation by isolated hepatocytes incubated with cysteine or methionine was not inhibited by the addition of 80 mM ethanol. A significantly decreased rate of GSH accumulation was, however, found in hepatocytes isolated from ethanol-intoxicated animals. Neither the in vivo pretreatment with ethanol nor its in vitro addition to isolated hepatocytes affected the rate of GSH efflux. The results suggest that ethanol itself, rather than its metabolic products, causes depletion of liver GSH, and that events occurring in vivo are required for such an effect to be exerted.