Electrophoresis of freshly prepared tissue homogenates of the Syrian golden hamster (Mesocricetus auratus) on starch gel followed by activity staining with ethanol as the substrate revealed three major alcohol dehydrogenase (ADH) isozymes. One of these isozymes, TT-ADH, found only in the testes of golden hamsters was previously purified and partially characterized (Keung WM: Biochem. Biophys. Res. Commun. 156:38-45, 1988). The other two, AA- and BB-ADH, which are most abundant in the liver, have now been purified by affinity chromatography on 4-(3-(N-(6-aminocaproyl)amino)propyl)pyrazole-sepharose and testosterone-17 beta-hemisuccinate-agarose. Hamster AA-, BB-, and TT-ADH are all homodimers of molecular weight near 80,000 and each contains 4 atoms of zinc. Amino acid analyses show that BB-ADH is most closely related to the gamma-form of human class I ADH, whereas AA- and TT-ADH are most closely related to the beta-form of the human enzyme. BB-ADH is the only hamster ADH that is active toward sterols and sensitive to testosterone and isoflavone inhibition. These results suggest that hamster BB- and human gamma gamma-ADH also share similar catalytic properties. AA- and TT-ADH are neither active toward sterols nor sensitive to testosterone or isoflavone inhibition; thus, they are functionally different from the human alpha alpha- or gamma gamma-ADHs. Compared with AA- and BB-ADH, TT-ADH exhibits much higher Km values toward primary aliphatic alcohols and cyclohexanol. AA- and BB-ADH share similar substrate specificities toward primary aliphatic alcohols. However, they exhibit different stereospecificities for secondary alcohols. BB-ADH prefers the (R)-(-)-isomer of 2-butanol, whereas AA-ADH prefers the (S)-(-)-isomer. These results further demonstrate that catalytically, hamster BB- and AA-ADH belong to different subfamilies of class I ADH.