In a previous study (Wu and Cederbaum, J. Biol. Chem. 271:23914-23919, 1996), ethanol was shown to be cytotoxic to HepG2 cells, which were transduced to express human cytochrome P-4502E1 (CYP2E1) but not to control HepG2 cells. The goal of the current study was to evaluate whether this toxicity was apoptotic in nature. Incubation of CYP2E1-expressing HepG2 cells with 100 mM ethanol for 2 days produced morphological changes and DNA fragmentation (in situ labeling, flow cytometry, and DNA ladder formation) indicative of apoptosis. No changes were observed in the control HepG2 cells that do not express CYP2E1. Ethanol-induced apoptosis was also observed in HepG2 cells transiently transfected to express CYP2E1. The ethanol-induced apoptosis was prevented by 4-methylpyrazole, an inhibitor of ethanol oxidation by CYP2E1, and by trolox, an antioxidant that prevents lipid peroxidation. Ethanol treatment of the cells expressing CYP2E1 resulted in increased activities of caspases 1 and 3. An inhibitor of these caspases prevented the ethanol-induced apoptosis in the stable cell lines and the transiently transfected cell lines. Ethanol did not cause apoptosis in a HepG2 cell line overexpressing bcl-2 plus CYP2E1, but did cause apoptosis in cell lines expressing CYP2E1 in the absence of bcl-2. These experiments demonstrate that ethanol can produce apoptosis in HepG2 cells that express CYP2E1. Increased production of reactive oxygen species and lipid peroxidation can be associated with apoptotic cell death. The prevention of the ethanol-induced apoptosis by 4-methylpyrazole and by trolox suggests that production of a prooxidative state as a consequence of ethanol oxidation by CYP2E1 results in eventual activation of caspases such as caspases 1 and 3, which can trigger the apoptotic process.