A number of recent reports indicate that massive overdoses of acetaminophen can produce a fulminant hepatic necrosis in man. Acetaminophen-induced hepatic necrosis thereforne was examised in experimental animals. Centrilobular hepatic necrosis similar to that seen in man was shown to be dose dependent in mice and rats. To determine whether acetaminophen-induced hepatotoxicity was mediated throug an active metabolite, acetaminophen concentrations in rat and mouse tissues were compared with the severity of liver damage after alteration of the rate of metabolism of acetaminophen. Phenobarbital pretreatment stimulated the disappearance of acetaminophen from tissues but markedly potentiated the hepatic necrosis. In contrast, piperonyl butoxide pretreatment inhibited the metabolism and disappearance of acetaminophen from tissues yet dramatically protected against hepatic necrosis. Additional studies demonstrated that pretreatment with 3-methylcholanthrene also potentiated acetaminophen-induced hepatic necrosis whereas cobaltous chloride, which inhibits synthesis of cytochrome P-450, prevented the hepatic damage. We propose that acetaminophen-induced hepatic necrosis is mediated by a toxic metabolite of acetaminophen.