beta-hexosaminidase is an essential lysosomal enzyme whose absence in man results in a group of disorders, the G(M2) gangliosidoses. beta-hexosaminidase activity is many times higher in the epididymis than in other tissues, is present in sperm, and is postulated to be required for mammalian fertilization. To better understand which cells are responsible for beta-hexosaminidase expression and how it is regulated in the male reproductive system, we quantitated the mRNA expression of the alpha- and beta-subunits of beta-hexosaminidase and carried out immunocytochemical localization studies of the enzyme in the rat testis and epididymis. beta-hexosaminidase alpha-subunit mRNA was abundant and differentially expressed in the adult rat testis and epididymis, at 13- and 2-fold brain levels, respectively. In contrast, beta-subunit mRNA levels in the testis and epididymis were 0.3- and 5-fold brain levels. During testis development from 7-91 postnatal days of age, testis levels of alpha-subunit mRNA increased 10-fold and coincided with the appearance of spermatocytes and spermatids in the epithelium; in contrast, beta-subunit mRNA was expressed at low levels throughout tests development. In isolated male germ cells, beta-hexosaminidase alpha-subunit expression was most abundant in haploid round spermatids, whereas the beta-subunit mRNA was not detected in germ cells. Within the epididymis both alpha- and beta-subunit mRNA concentrations were highest in the corpus, with 1.5-fold and 9-fold initial segment values, respectively. Light microscopic immunocytochemistry revealed that beta-hexosaminidase was localized to Sertoli cells and interstitial macrophages in the testis. In the epididymis, beta-hexosaminidase staining was most intense in narrow cells in the initial segment, principal cells in the caput and proximal corpus, and clear cells throughout the duct. Electron microscopic immunocytochemistry revealed that beta-hexosaminidase was predominantly present in lysosomes in Sertoli and epididymal cells. The cellular and regional specificity of beta-hexosaminidase immunolocalization suggest an important role for the enzyme in testicular and epididymal functions.