Background: Although oxidative stress and deficits in hepatic energy metabolism have been implicated as important factors in the initiation of alcoholic liver disease, their relative contribution to ethanol-induced cell death is not known. The purpose of this study was to examine the effects of chronic ethanol administration on hepatocyte reactive oxygen species (ROS) generation, energy state, and viability, as well as the effect of dietary fat on these parameters. Methods: Male Sprague-Dawley rats were fed liquid diets that provided 36% total calories as ethanol, with fat as either 12% (low fat) or 35% (high fat) of total calories. Pair-fed controls received liquid diets in which maltose-dextrin was substituted for ethanol calories. The fluorescent probe 2',7'-dichlorofluorescin diacetate was used to detect Res, lactate dehydrogenase leakage was used to assess viability, and ATP levels were used as a measure of the energy state. The effect of chronic ethanol feeding on these parameters was determined by incubating hepatocytes under a 5% oxygen-containing atmosphere or an atmosphere ≤ 1% oxygen for 60 min. Results: In general, chronic ethanol feeding stimulated Res production and decreased ATP concentrations, which were associated with decreased viability in hepatocytes isolated from rats fed either high- or low-fat, ethanol- containing diets, compared to the corresponding controls. Incubation under an atmosphere ≤ 1% oxygen and/or ethanol (10 mM) augmented these effects in both high- and low-fat control and ethanol-fed hepatocytes. The addition of antimycin to the incubations increased Res production, decreased ATP concentrations, and accelerated loss of hepatocyte viability. Viability loss under all conditions used in this study was correlated with decreases in cellular ATP. Conclusions: Comparisons of incubations performed under the two oxygenation conditions revealed that viability loss was inversely associated with Res production, which indicates that ATP loss and not Res production was a better predictor of loss in cell integrity. This study also demonstrates that the level of dietary fat has only minor effects on generation of Res and the cellular energy state. In contrast, ethanol consumption had significant effects on generation of Res, energy state, and hepatocyte viability.