The possible participation of acute oxidative stress in the in vivo mechanism by which acetaminophen (APAP) induces hepatocellular injury was examined. Male Sprague-Dawley rats were administered 3-methylcholanthrene, fasted for 18 h, then given APAP and sacrificed after a further 6 h of fasting. Extensive centrilobular liver cell necrosis along with markedly elevated serum activity of aminotransferases was observed. Liposome-encapsulated human recombinant Cu-Zn superoxide dismutase (LSOD) administered 1 or 0.5 h prior to APAP or simultaneously with the toxin completely prevented APAP-induced hepatocellular injury. In contrast, LSOD administered 5 or 2.5 h before or 1, 2.5 or 5 h after the toxin treatment did not prevent APAP toxicity. Incomplete protection against APAP-induced injury was obtained when LSOD was administered 0.5 h after the toxin. These results support the proposal of an oxidative mechanism for APAP hepatotoxicity.