Livers from rats fasted for 24 hr were subjected to nonrecirculating perfusion with Krebs-Ringer bicarbonate solution containing 10 mM ethanol. The deuterium isotope effect was measured using (1-R)-[1-14C,1-2H]ethanol. A value of 2.57 +/- 0.09 (SD) was obtained independent of the direction of perfusion. Oxygen uptake and ethanol metabolism in contrast were significantly increased when reverse perfusion (i.e., from vena cava to vena portae) was used. The magnitude of the isotope effect indicates that contribution from microsomal ethanol-oxidizing system if this is the only supplementary system is 9.8% under the experimental conditions. At high ethanol concentrations, the contribution would approach 18%. Equal activities of microsomal ethanol-oxidizing system and catalase under the experimental conditions would mean that both contribute 7.3% of the total ethanol metabolism. At high ethanol concentrations (80 mM), however, catalase will be 6.8% and microsomal ethanol-oxidizing system is calculated to 13.3%. Preliminary experiments with rats pretreated with phenobarbital showed no change in the isotope effect or in the rate of ethanol metabolism, but a 40-50% increase in oxygen consumption. The acetaldehyde concentration in the effluent medium was below 1 microM.