Abstract In isolated hepatocytes from fed and starved rats, basal rates of ethanol oxidation were 1.15 and 0.71 μmoles/g wet wt, respectively, and were unchanged over the ethanol concentration range 8–96 mM. The addition of 4-methyl pyrazole (4 mM), a competitive inhibitor of alcohol dehydrogenase, largely abolished ethanol oxidation from 8mM ethanol, while at an ethanol concentration of 96 mM, the oxidation rate was inhibited by 87 per cent. Pyrazole was a less effective inhibitor of alcohol oxidation than 4-methyl pyrazole. In hepatocytes isolated from rats treated with ethanol, phenoharbitone or 3-amino-triazole, basal rates of ethanol oxidation were the same at ethanol concentrations of 8–96 mM and the rates were similar to, and never exceeded, the rate found in hepatocytes from normal fed rats. 4-Methyl pyrazole inhibited ethanol oxidation to the same extent in all liver cell preparations. regardless of the treatment the donor animal had received. Pyruvate stimulated cellular ethanol oxidation irrespective of the prior treatment of the donor animal. This stimulation, together with the ethanol-induced accumulation of lactate, was abolished by 4-methyl pyrazole. This suggests that the capacity for alcohol oxidation in isolated liver cells is generally limited by the lack of suitable acceptors for the hydrogen generated in the cytoplasm by the alcohol dehydrogenase-catalysed oxidation of ethanol to acetaldehyde. Methylene blue, phenazine methosulphate and menadione stimulated both ethanol oxidation and respiration, irrespective of the prior treatment of the donor animal. This enhancement of ethanol oxidation and respiration was prevented by 4-methyl pyrazole. These artificial electron acceptors appear to act by circumventing normal pathways for the oxidation of cytoplasmic NADH generated in the conversion of ethanol to acetaldehyde. In cells from each treatment group, antimycin was more effective than rotenone as an inhibitor of ethanol oxidation; inhibition of ATP formation by oligomycin had least effect on alcohol oxidation. Ethanol oxidation by cells from alcohol-treated rats was most affected by these inhibitors of mitochondrial respiration. These results indicate that under a wide variety of experimental conditions the contribution of the postulated microsomal ethanol oxidizing system to ethanol oxidation in isolated, intact liver cells appears minimal. Thus they cast doubt on a physiological role for this system in vivo.