The mechanisms underlying hepatocyte sensitization to tumor necrosis factor- (TNF-)-mediated cell death remain unclear. Increases in hepatocellular oxidant stress such as those that occur with hepatic overexpression of cytochrome P-450 2E1 (CYP2E1) may promote TNF- death. TNF- treatment of hepatocyte cell lines with differential CYP2E1 expression demonstrated that overexpression of CYP2E1 converted the hepatocyte TNF- response from proliferation to apoptotic and necrotic cell death. Death occurred despite the presence of increased levels of nuclear factor-B transcriptional activity and was associated with increased lipid peroxidation and GSH depletion. CYP2E1-overexpressing hepatocytes had increased basal and TNF--induced levels of c-Jun NH₂-terminal kinase (JNK) activity, as well as prolonged JNK activation after TNF- stimulation. Sensitization to TNF--induced cell death by CYP2E1 overexpression was inhibited by antioxidants or adenoviral expression of a dominant-negative c-Jun. Increased CYP2E1 expression sensitized hepatocytes to TNF- toxicity mediated by c-Jun and overwhelming oxidative stress. The chronic increase in intracellular oxidant stress created by CYP2E1 overexpression may serve as a mechanism by which hepatocytes are sensitized to TNF- toxicity in liver disease.