The molecular basis of the alcohol dehydrogenase (ADH)-negative deer mouse (Peromyscus maniculatus) has been investigated. Several classes of mammalian ADHs have been recognized based upon biochemical and structural properties. ADH cDNA clones identified by hybridization to a mouse class I ADH cDNA clone were obtained from a deer mouse ADH-positive liver cDNA library. This cDNA has been identified as being a class I sequence and represents the deer mouse Adh-1 gene. An additional cDNA sequence identified in both the ADH-positive and -negative deer mouse cDNA libraries was identified by weak cross-hybridization to the mouse cDNA. This cDNA encodes an amino acid sequence representing a new class of mammalian ADH, and the deer mouse gene for this ADH is named Adh-2. ADH-negative deer mice do not produce mRNA, that is detected by the Adh-1 cDNA probe. However, both stocks of deer mice produce high levels of Adh-2 mRNA in liver. Southern analysis using an essentially full-length Adh-1 cDNA probe has shown that the Adh-1 gene is deleted in the ADH-negative mice. Biochemical analysis of enzyme activity suggests at least three ADH polypeptides are expressed in different tissues and have somewhat different substrate specificities, as in the mouse.