In a few patients diclofenac produces mild increases in serum aminotransferase activity and in rare cases may be associated with the occurrence of fulminant hepatic necrosis. Both direct toxic effects of a diclofenac metabolite and hypersensitivity reactions have been suggested as possible molecular mechanisms of liver injury. We investigated the pathways of bioactivation and cytotoxicity of diclofenac, which undergoes both aromatic hydroxylation and acyl glucuronidation, in short-term cultured rat hepatocytes. LDH release was first evident after 4 hr of incubation with diclofenac (> 500 microM). In addition, time- and concentration-dependent covalent binding of [14C]diclofenac to hepatocellular proteins occurred, indicating the presence of a reactive intermediate. To specifically explore the role of the acyl glucuronidation pathway in the induction of cytotoxicity and covalent drug-protein adducts, we used two inhibitors of the UDP-glucuronosyltransferase (UDPGT), borneol and 7,7,7-triphenylheptyl-UDP. LDH release was markedly increased in the presence of either UDPGT inhibitor. Alternatively, covalent binding to hepatocellular proteins was greatly reduced when the glucuronide formation was selectively blocked. Furthermore, in vitro inhibition of P450-dependent oxidative biotransformation with the selective inhibitor of the CYP2C subfamily sulfaphenazole or with cimetidine markedly reduced the extent of cytotoxicity, whereas the degree of covalent adduct formation remained unchanged. Similarly, pretreatment of the rats with phenobarbital (80 mg/kg/day for 4 days) delayed the onset and reduced the extent of diclofenac-induced LDH release. Collectively, these results indicate that the formation of a toxic diclofenac metabolite(s) catalyzed by P4502C in hepatocytes leads to acute lethal cell injury, whereas diclofenac acyl glucuronide formation is associated with covalent binding of a reactive metabolite to hepatocellular proteins that is not related to the acute cytotoxicity. The protein adduct formation and its modulation by UDPGT may, however, be toxicologically relevant for the expression of diclofenac hepatitis.