cDNAs coding for class II alcohol dehydrogenase were isolated from a rabbit-liver cDNA library. Deduced amino acid sequences show that isozymic forms of rabbit class II alcohol dehydrogenase exist, with a positional identity of 88.4%. A high variability in structure of class II alcohol dehydrogenase between the species is also reflected in function. The rabbit II-1 isozyme shows common characteristics with the human enzyme, but has a lower Km value for ethanol, 4.2 mM. The II-2 isozyme shows restriction for aliphatic alcohols longer than pentanol. For shorter alcohols the II-2 form has similar Km values as the II-1 isozyme, 5.5 mM for ethanol, but is a low activity variant with a 10-fold decrease in k(cat) values compared with II-1. Nevertheless, II-2 has a higher specificity for benzoquinone than II-1 due to a lower Km value, 80 microM compared with 1 mM, and is in this sense more like the human class II enzyme. In addition a rabbit class III alcohol dehydrogenase cDNA was isolated that encodes a typical class III enzyme/glutathione-dependent formaldehyde dehydrogenase. The finding of isozymic forms of class II alcohol dehydrogenase is in line with the evolution of the system of medium-chain alcohol dehydrogenases with different enzymes, different classes and different isozymes and further underline the complexity of the entire mammalian alcohol dehydrogenase system.