Alcohol-fed hamsters were used to study the mechanism by which acetaminophen initiates hepatotoxicity. Animals maintained on an ethanol-containing diet (Group B) exhibited an increased mortality rate after administration of acetaminophen (400 mg/kg) as compared to control hamsters (Group A). However, in those animals in which the ethanol-containing diet had been replaced by the control diet 24 hr before receiving acetaminophen (Group C), significant protection against acetaminophen toxicity was observed as compared to control animals (Group A). This observation correlates well with the finding that Group C hamsters had higher levels of glutathione and catalase than was found in either Group A or Group B animals. It was also demonstrated that acetaminophen was oxidized by cytochrome P-450, producing acetaminophen free radical and hydrogen peroxide. The free radical in the presence of oxygen was found to generate superoxide and presumably N-acetyl-p-benzoquinone imine. Microsomal lipid peroxidation was found to be stimulated markedly in the presence of acetaminophen. The role of glutathione in protecting hamsters from acetaminophen-mediated hepatotoxicity is discussed.