Our laboratory has previously reported on the ability of 4-hydroxynonenal (4-HNE), a primary product of lipid peroxidation, to inhibit acetaldehyde metabolism in isolated mouse liver mitochondria. The purpose of the present study is to determine whether the co-metabolism of ethanol and 4-HNE compromises the elimination of either substrate in isolated rat hepatocytes. Hepatocytes were isolated and incubated with ethanol and 4-HNE. Ethanol elimination and acetaldehyde accumulation were monitored by gas chromatography, whereas 4-HNE elimination and metabolite accumulation were measured by UV detection and reversed-phase HPLC at 202 nm. In the absence of 4-HNE, hepatocytes metabolized ethanol at an initial rate of 9.4 nmol/min/million cells. Ethanol elimination was moderately inhibited by the presence of 4-HNE. Accumulation of ethanol-derived acetaldehyde was not apparent in incubations with only ethanol. In contrast, in incubations containing both substrates, ethanol-derived acetaldehyde accumulation exceeded that observed in hepatocytes exposed only to ethanol and was proportional to the 4-HNE concentration in the incubations. In all instances, the rate of 4-HNE elimination was not compromised by the presence of ethanol. Accordingly, ethanol metabolism did not alter the oxidative or conjugative metabolism of 4-HNE. However, the reductive metabolism of 4-HNE was affected by the presence of ethanol, wherein accumulation of 1,4-dihydroxy-2-nonene increased > 2-fold of that observed in incubations with only 4-HNE. To determine further if 4-HNE and ethanol are metabolized through the same metabolic pathways, cells were preincubated with either 4-methylpyrazole or cyanamide to inhibit alcohol dehydrogenase (E.C. 1.1.1.1.) and aldehyde dehydrogenase (E.C. 1.2.1.2.), respectively. Expectantly, 4-methylpyrazole blocked the formation of 1,4-dihydroxy-2-nonene, but had no effect on the rate of 4-HNE elimination. In contrast, cyanamide substantially inhibited the formation of 4-hydroxy-2-nonenoic acid, decreased the rate of 1,4-dihydroxy-2-nonene formation, but did not decrease the elimination rate of 4-HNE. Overall, these results support our previous observation that 4-HNE inhibits acetaldehyde metabolism and establish that ethanol and 4-HNE are metabolized through the same alcohol dehydrogenase- and aldehyde dehydrogenase-mediated pathways. These data continue to suggest that, as a consequence of enhanced lipid peroxidation resulting from chronic ethanol consumption, increased 4-HNE levels could compromise cellular elimination of ethanol-derived acetaldehyde and thus function in the potentiation of alcoholic liver fibrosis.