An affinity-purified polyclonal antibody against N-acetyl-beta-D-hexosaminidase (EC 3.2.1.52) from adult rat epididymis was used to develop an ELISA, to localize tissue antigen by immunocytochemistry, and to identify isoforms of the enzyme by Western blot analysis. While peak activity level was measured in the corpus region of the epididymis, the quantity of soluble enzyme protein was highest in the caput region. Luminal caput sperm were intensely immunopositive, whereas epithelial cells were weakly stained. The enzyme was localized to epithelial and sperm cells of the corpus and caudal regions. In the testis, the enzyme was restricted primarily to lymphatic spaces. Testicular cells were unreactive, but spermatids were weakly stained. Western blot analysis revealed three prominent immune-reactive protein variants of approximately 63 kDa, approximately 55 kDa, and approximately 31 kDa. The approximately 31-kDa immune-reactive protein variant was reduced by > 84% in the caput epididymis compared to the cauda and corpus regions. These data suggest that regional differences in N-acetyl-beta-D-hexosaminidase activity in the rat epididymis may be due to differences in cellular synthesis and/or posttranslational processing of the enzyme.