Although the AdhN/AdhN strain of Peromyscus maniculatus (so-called ADH- deermouse) has been previously considered to be deficient in ADH, we found ADH isozymes of Classes II and III but not Class I in the liver of this strain. On the other hand, the AdhF/AdhF strain (so-called ADH+ deermouse), which has liver ADH activity, had Class I and III but not Class II ADH in the liver. In the stomach, Class III and IV ADHs were detected in both deermouse strains, as well as in the ddY mouse, which has the normal mammalian ADH system with four classes of ADH. These ADH isozymes were identified as electrophoretic phenotypes on the basis of their substrate specificity, pyrazole sensitivity, and immunoreactivity. Liver ADH activity of the ADH- strain was barely detectable in a conventional ADH assay using 15 mM ethanol as substrate; however, it increased markedly with high concentrations of ethanol (up to 3 M) or hexenol (7 mM). Furthermore, in a hydrophobic reaction medium containing 1.0 M t-butanol, liver ADH activity of this strain at low concentrations of ethanol (< 100 mM) greatly increased (about sevenfold), to more than 50% that of ADH+ deermouse. These results were attributable to the presence of Class III ADH and the absence of Class I ADH in the liver of ADH- deermouse. It was also found that even the ADH+ strain has low liver ADH activity (< 40% that of the ddY mouse) with 15 mM ethanol as substrate, probably due to low activity in Class I ADH. Consequently, liver ADH activity of this strain was lower than its stomach ADH activity, in contrast with the ddY mouse, whose ADH activity was much higher in the liver than in the stomach, as well as other mammals. Thus, the ADH systems in both ADH- and ADH+ deermouse were different not only from each other but also from that in the ddY mouse; the ADH- strain was deficient in only Class I ADH, and the ADH+ strain was deficient in Class II ADH and down-regulated in Class I ADH activity. Therefore, Class III ADH, which was found in both strains and activated allosterically, may participate in alcohol metabolism in deermouse, especially in the ADH- strain.