Experiments were designed to test whether the protective effect of N-acetylcysteine against acetaminophen hepatotoxicity precedes arylation of tissue or whether protection occurs after arylation of tissue. Investigation of potential postarylation actions showed that N-acetylcysteine was unable to attenuate the liver necrosis caused by acetaminophen or several other hepatotoxins that act through chemically reactive metabolites. Furthermore, varying the time and route of N-acetylcysteine treatment indicated that the late protection against acetaminophen mortality probably was a consequence of pharmacokinetic factors rather than postarylation intervention in the process of cell death. The antidote was found to inhibit covalent binding of acetaminophen by about 70% when N-acetylcysteine protected against liver necrosis. Treatment regimens that had no effect upon covalent binding also had no effect on acetaminophen hepatotoxicity. Previous failures to detect this relationship apparently occurred because of a failure to consider biological events important in the pathophysiology of acetaminophen-induced necrosis, particularly the marked intrahepatic hemorrhage and vascular congestion with liver engorgement by protein and fluid. These results support the hypothesis that sulfhydryl nucleophiles such as N-acetylcysteine act primarily through prearylation mechanisms to decrease the amount of reactive metabolite available for initiation of hepatic injury.