Alcohol dehydrogenase isozymes from mouse liver (A2 and B2) and stomach (C2) tissues have been purified to homogeneity using triazine-dye affinity chromatography. The enzymes are dimers with similar but distinct subunit sizes, as determined by SDS/polyacrylamide gel electrophoresis: A, 43000; B, 39000, and C, 47000. Zinc analyses and 1,10-phenanthroline inhibition studies indicated that the A and C subunits each contained two atoms of zinc, with at least one being involved catalytically, whereas the B subunit probably contained a single non-catalytic zinc atom. The isozymes exhibited widely divergent kinetic characteristics. A2 exhibited a Km value for ethanol of 0.15 mM and a broad substrate specificity, with Km values decreasing dramatically with an increase in chain length; C2 also exhibited this broad specificity for alcohols but showed a Km value of 232 mM for ethanol. These isozymes also showed broad substrate specificities as aldehyde reductases. In contrast, B2 showed no detectable activity as an aldehyde reductase for the aldehydes examined, and used ethanol as substrate only at very high concentrations (greater than 0.5 M). The isozyme exhibited low Km and high Vmax values, however, with medium-chain alcohols. Immunological studies showed that A2 was immunologically distinct from the B2 and C2 isozymes. In vitro molecular hybridization studies gave no evidence for association between the alcohol dehydrogenase subunits. The results confirm genetic analyses [Holmes, Albanese, Whitehead and Duley (1981) J. Exp. Zool. 215, 151-157] which are consistent with at least three structural genes encoding alcohol dehydrogenase in the mouse and confirm the role of the major liver isozyme (A2) in ethanol metabolism.