The vitamin A metabolite retinoic acid plays an essential signaling role in spermatogenesis by acting as a ligand for nuclear retinoic acid receptors. However, little is known about the regulation of retinoic acid synthesis from vitamin A (retinol). Here we have examined mouse testis and epididymis for the presence of endogenous retinoic acid and for the expression of genes encoding class I and class IV alcohol dehydrogenases (ADH), both of which catalyze retinol oxidation, the rate-limiting step in the conversion of retinol to retinoic acid. Using a bioassay we found that mouse testis and epididymis both have significant levels of retinoic acid ranging from 7 to 8 pmol/g, an amount known to be sufficient to optimally activate retinoic acid receptors. In situ hybridization analysis of mouse testis revealed that class I ADH mRNA was localized in Sertoli cells and Leydig cells, while class IV ADH mRNA was confined to late spermatids. In the epididymis, class I ADH mRNA was detected in both principal and basal cells, whereas class IV ADH mRNA was limited to basal cells. Immunohistochemical analyses of testis indicated that class I ADH protein was localized in Sertoli and Leydig cells, whereas class IV ADH protein was observed only in late spermatids. Class I ADH protein was localized in principal and basal cells of the cauda epididymidis but only in basal cells of the caput epididymidis. Class IV ADH protein was limited to basal cells along the entire length of the epididymis. These results support a role for ADHs during spermatogenesis, potentially as retinol dehydrogenases catalyzing local retinoic acid synthesis in the testis and epididymis.