A class II type alcohol dehydrogenase from rat liver was characterized at the cDNA level after screening cDNA libraries in combination with PCR amplification of the 5'-part. The open reading frame translates into a polypeptide of 376 amino acid residues, which show 73% positional identity to the human class II enzyme. This suggests that the class II enzyme is the most variable form of the mammalian alcohol dehydrogenases. A deletion is apparent corresponding to position 294 of the human enzyme and amino acid residues unique to the rat protein of those interacting with the coenzyme NAD+ are found at positions 47, 51, 178, and 271. Position 47 is occupied by Pro instead of Arg or His found in most mammalian alcohol dehydrogenases. This exchanged residue will not hydrogen bond to the pyrophosphate of the coenzyme and will change the local environment around position 47 to strictly hydrophobic.