The relative contributions to ethanol metabolism of extrahepatic alcohol dehydrogenase (ADH) and of liver microsomes were assessed in deermice, which lack hepatic low Km ADH (ADH-). In vitro kinetic studies showed the existence of high Km (> 1 M) ADH activity in the liver and kidney, and an enzyme with intermediate Km in the gastric mucosa (Km = 133 mM), whereas the low Km ADH was missing. With deuterated ethanol, ADH- deermice showed a significant exchange of reducing equivalents that had been equated with ethanol metabolism by others, whereas we found a poor correlation between the rate of exchange and the rate of metabolism. In vitro studies with subcellular fractions, isolated hepatocytes, and tissue slices revealed that neither liver, nor kidney, nor stomach from ADH- deermice contributed to exchange of reducing equivalents. These findings clearly indicated that the ADHs with high or intermediate Km of the tissues studied are not responsible for the exchange. Furthermore, gastrectomized ADH- deermice still showed an exchange of reducing equivalents, thereby dissociating exchange from gastric ADH activity. Moreover, pretreatment with cimetidine (50 mg/kg body weight), an inhibitor of gastric ADH, did not alter the rate of total ethanol elimination when ethanol was given intraperitoneally. In conclusion, when ethanol was given parenterally, the microsomal ethanol-oxidizing system rather than gastric ADH is a major pathway of ethanol oxidation in ADH- deermice, whereas both pathways contribute significantly to the metabolism of orally administered ethanol.