The hepatotoxicity of acetaminophen (APAP) overdose depends on metabolic activation to a toxic reactive metabolite via hepatic mixed function oxidase. In vitro studies have indicated that APAP may also be cooxidized by prostaglandin H synthetase. The present experiments were designed to assess the possible contribution of hepatic prostaglandin synthesis to APAP toxicity. Adult fed male mice were overdosed with 400 mg APAP/kg. Liver toxicity was estimated by measurement of serum transaminases. Hypertonic xylitol or sodium chloride (2250 mOsm/l), administered intragastrically to stimulate prostaglandin synthesis, increased APAP toxicity. By contrast, the cyclooxygenase inhibiting drugs aspirin (at 25 mg/kg) and indomethacin (at 10 mg/kg) protected against APAP-induced toxicity. APAP kinetics were not affected by hypertonic xylitol or indomethacin, nor were hepatic glutathione levels in overdosed mice. Imidazole, a nonspecific thromboxane synthetase inhibitor, also protected overdosed mice. This drug prolonged hexobarbital sleeping time and prevented the depletion of hepatic glutathione that followed APAP intoxication. Thus, the data support the conclusion that APAP-induced hepatoxicity may be modulated not only by inhibition of cytochrome P450 mediated oxidation, but also by controlling hepatic cyclooxygenase activity.