Increased expression of cytochrome P450 2E1 (CYP2E1) occurs in alcoholic liver disease, and leads to the hepatocellular generation of toxic reactive oxygen intermediates (ROI). Oxidative stress created by CYP2E1 overexpression may promote liver cell injury by sensitizing hepatocytes to oxidant-induced damage from Kupffer cell-produced ROI or cytokines. To determine the effect of CYP2E1 expression on the hepatocellular response to injury, stably transfected hepatocytes expressing increased (S-CYP15) and decreased (AN-CYP10) levels of CYP2E1 were generated from the rat hepatocyte line RALA255-10G. S-CYP15 cells had increased levels of CYP2E1 as demonstrated by Northern blot analysis, immunoblotting, catalytic activity, and increased cell sensitivity to death from acetaminophen. Death in S-CYP15 cells was significantly decreased relative to that in AN-CYP10 cells following treatment with hydrogen peroxide and the superoxide generator menadione. S-CYP15 cells underwent apoptosis in response to these ROI, whereas AN-CYP10 cells died by necrosis. This differential sensitivity to ROI-induced cell death was partly explained by markedly decreased levels of glutathione (GSH) in AN-CYP10 cells. However, chemically induced GSH depletion triggered cell death in S-CYP15 but not AN-CYP10 cells. Increased expression of CYP2E1 conferred hepatocyte resistance to ROI-induced cytotoxicity, which was mediated in part by GSH. However, CYP2E1 overexpression left cells vulnerable to death from GSH depletion.