Chromosomal DNA samples derived from various primates and other mammals (horse, sheep, rabbit, and mouse) were digested with restriction endonuclease and hybridized with a probe of the sixth exon of the human ADH gene, which is highly conserved in the class I alcohol dehydrogenase of these mammalian species. The copy number of the class I ADH gene in each species was estimated from the number of hybridized bands. Primate DNA samples showed three distinct bands in the blots of PstI digest and DraI digest. Moreover, most of the bands from primate DNA showed a similarity in size so as to allow us to assign the ADH1, ADH2, and ADH3 homologues in each species. In contrast, mouse has only one gene, and rabbit, sheep, and horse seem to have only two genes, for the class I ADH, which showed divergent hybridization bands. These results are consistent with the view that the human class I ADH gene cluster has been generated through gene multiplication events which occurred before the Catarrhini branch point in the course of primate evolution.