Humans possess five classes of alcohol dehydrogenase (ADH), including forms able to oxidize ethanol or formaldehyde as part of a defense mechanism, as well as forms acting as retinol dehydrogenases in the synthesis of the regulatory ligand retinoic acid. However, the mouse has previously been shown to possess only three forms of ADH. Hybridization analysis of mouse genomic DNA using cDNA probes specific for each of the five classes of human ADH has now indicated that mouse DNA cross-hybridizes to only classes I, III, and IV. With human class II or class V ADH cDNA probes, hybridization to mouse genomic DNA was very weak or undetectable, suggesting either a lack of these genes in the mouse or a high degree of mutational divergence relative to the human genes. cDNAs for murine ADH classes I and III have previously been cloned, and we now report the cloning of a full-length mouse class IV ADH cDNA. In Northern blot analyses, mouse class IV ADH mRNA was abundant in the stomach, eye, skin, and ovary, thus correlating with the expression pattern for the mouse Adh-3 gene previously determined by enzyme analysis. In situ hybridization studies on mouse stomach indicated that class IV ADH transcripts were abundant in the mucosal epithelium but absent from the muscular layer. Comparison of the expression patterns for all three mouse ADH genes indicated that class III was expressed ubiquitously, whereas classes I and IV were differentially expressed in an overlapping set of tissues that all contain a large component of epithelial cells. This expression pattern is consistent with the ability of classes I and IV to oxidize retinol for the synthesis of retinoic acid known to regulate epithelial cell differentiation. The results presented here indicate that the mouse has a simpler ADH gene family than the human but has conserved class IV ADH previously shown to be a very active retinol dehydrogenase in humans.