Doses of ³H-acetaminophen (300-750 mg/kg) that cause necrosis in mice were shown to result in large amounts of radiolabeled material covalently bound to mouse liver protein in vivo (2 nmol/mg of protein or approximately one molecule bound per two molecules of protein in microsomes and cytoplasm). Both covalent binding and hepatic necrosis were dose dependent, and the peak level of binding preceded the development of recognizable necrosis by at least one to two hours. Pretreatment of mice with phenobarbital, piperonyl butoxide or cobaltous chloride, which changed the rate of metabolism of ³H-acetaminophen and altered the severity of hepatic necrosis, similarly affected the extent of hepatic binding of radiolabeled metabolite. Furthermore, the degree of binding in individual mice was always directly proportional to the severity of hepatic necrosis regardless of the biologic variation among various animals. Accordingly, we propose that acetaminophen-induced hepatic necrosis may be caused by the covalent binding of a chemically reactive metabolite to vital hepatsic macromolecules.