The aim of this study was to investigate the hepatocellular site of reactive oxygen species generation during acute ethanol metabolism. Reactive oxygen species production was detected using the 2',7'-dichlorofluorescein fluorescence assay and cell injury was determined by lactate dehydrogenase release. Incubation with 1 and 10 mM ethanol increased the production of reactive oxygen species by 72% and 151%, respectively, which was associated with mild decreases in cell viability. Antimycin, a mitochondrial complex III inhibitor, elicited a 17-fold increase in the levels of reactive oxygen species and markedly decreased hepatocyte viability and ATP levels. Ethanol increased reactive oxygen species production and the cytosolic NADH/NAD+ ratio in antimycin-treated cells. Rotenone, a mitochondrial complex I inhibitor that allows electron flow through the flavin mononucleotide (FMN), but prevents electron flow to complex III, significantly increased reactive oxygen species production in untreated cells, but decreased reactive oxygen species production in antimycin plus ethanol-treated cells. Diphenyliodonium, a mitochondrial complex I inhibitor that inhibits electron flow through FMN, attenuated reactive oxygen species generation in all groups. Fructose prevented cytotoxicity in all treatment groups. Though they do not eliminate the participation of other intracellular compartments, these results indicate that the NADH dehydrogenase complex, as well as complex III of mitochondria, are involved in ethanol-related production of reactive oxygen species.