Mitochondria are thought to play a major role in hepatic oxidative stress associated with alcohol-induced liver injury. Thus, the hypothesis that delivery of the mitochondrial isoform of superoxide dismutase (Mn-SOD) via recombinant adenovirus would reduce alcohol-induced liver injury was tested. Rats were given recombinant adenovirus containing Mn-SOD (Ad.SOD2) or -galactosidase (Ad.lacZ), and then fed alcohol enterally for four weeks. Superoxide dismutase expression and activity of Ad.SOD2 in liver mitochondria of infected animals was increased nearly 3-fold compared to Ad.lacZ infected controls. Mitochondrial glutathione levels in Ad.lacZ infected animals were decreased after four weeks of chronic ethanol as expected, but were unchanged in Ad.SOD2 infected animals. Alanine aminotransferase was elevated significantly by ethanol, an effect that was prevented by Ad.SOD2. Moreover, pathology (e.g., the sum of steotosis, inflammation, and necrosis) was elevated dramatically by ethanol in Ad.lacZ treated rats. This effect was also blunted in animals infected with Ad.SOD2. Neutrophil infiltration was increased about 3-fold in livers from both Ad.lacZ and Ad.SOD2 infected rats by ethanol treatment. Moreover, ESR-detectable free radical adducts in bile were increased about 8-fold by ethanol. Using ¹³C-labeled ethanol, it was determined that nearly 60% of total adducts were due to the a-hydroxyethyl radical adduct. This increase in radical formation was blocked completely by Ad.SOD2 infection. Furthermore, apoptosis of hepatocytes was increased about 5-fold by ethanol, an effect also blocked by Ad.SOD2. Interestingly, TNF mRNA was elevated to the same extent in both Ad.lacZ- and Ad.SOD2-infected animals following ethanol exposure. These data suggest that hepatocyte mitochondrial oxidative stress is involved in alcohol-induced liver damage and likely follows Kupffer cell activation, cytokine production and neutrophil infiltration. These results also support the hypothesis that mitochondrial oxidant production is a critical factor in parenchymal cell death caused by alcohol.