The possibility that glutathione may protect against acetaminophen-induced hepatic necrosis was examined. Pretreatment of mice with diethyl maleate, which depletes hepatic glutathione, potentiated acetaminophen-induced hepatic necrosis, whereas pretreatment with cysteine, a glutathione precursor, prevented hepatic damage. Administration of acetaminophen caused a dose-dependent depletion of hepatic glutathione. Glutathione depletion by acetaminophen was enhanced by treatments that potentiate the hepatic necrosis and covalent binding produced by the toxic metabolite of acetaminophen. Conversely, glutathione depletion was inhibited by treatments that protect against these toxic effects. Moreover, covalent binding of this metabolite to hepatic macromolecules did not occur until the availability of glutathione was exhausted through conjugation with the metabolite. Similarly, alteration of glutathione availability by pretreatment with diethyl maleate or cysteine, respectively, increased or decreased covalent binding of the toxic metabolite. We propose that. a fundamental role of glutathione in the body may be to protect tissues against electrophilic attack by drug metabolites and other alkylating agents.