CYP2E1 induction by ethanol is one mechanism by which ethanol creates oxidative stress in the liver. The superoxide dismutases (SODs) are an important antioxidant enzyme defense system against reactive oxygen species (ROS). To investigate the protective role of SOD against CYP2E1–dependent toxicity, a transfected HepG2 cell line overexpressing CYP2E1 (E47 cells) was infected with adenoviral vectors containing Cu/Zn–SOD complementary DNA (cDNA) (Ad.SOD1) and Mn–SOD cDNA (Ad.SOD2). Forty–eight hours after infection, intracellular levels and activity of Cu/Zn–SOD and Mn–SOD were increased about 2– and 3–fold, respectively. Localization of the overexpressed Cu/Zn–SOD in the cytosol and Mn–SOD in the mitochondria was confirmed by assaying the levels and activity of SOD in the corresponding isolated fractions. Arachidonic acid (AA) plus iron–induced cell death was partially prevented in both Ad.SOD1– and Ad.SOD2–infected E47 cells. Overexpression of Cu/Zn–SOD and Mn–SOD also partially protected E47 cells from the increase in reactive oxygen production and lipid peroxidation and the loss of mitochondrial membrane potential induced by AA and iron. Infection with Cu/Zn–SOD and Mn–SOD also protected the E47 cells against AA toxicity or buthionine sulfoximine (BSO)–dependent toxicity. CYP2E1 levels and catalytic activity were not altered by overexpression of Cu/Zn–SOD or Mn–SOD. Cu/Zn–SOD in the cytosol and Mn–SOD in mitochondria each are capable of protecting HepG2 cells expressing CYP2E1 against cytotoxicity induced by pro–oxidants. In conclusion, these enzymes may be useful in the prevention or improvement of liver injury produced by agents known to be metabolized by CYP2E1 to reactive intermediates and to cause oxidative stress.