The purpose of the present study was to purify and kinetically characterize N-acetyl-beta-D-hexosaminidases A and B (EC 220.127.116.11) from the caput, corpus and caudal regions of the adult rat epididymis. The molecular mass of the purified native enzyme was approximately 250,000 and approximately 223,000 daltons for the A and B isozymes, with a subunit molecular mass of approximately 63,000 and approximately 56,000 daltons, as determined by size exclusion chromatography and gel electrophoresis under reducing conditions. The apparent Michaelis-Menten constant and maximum velocity values were 0.60, 1.55 and 0.68 mM and 0.54, 3.20 and 2.30 microM/min./mg protein for the enzyme purified from the caput, corpus and caudal regions, respectively. These values were determined by using p-nitrophenyl-N-acetyl-beta-D-glucosaminide as the substrate. These data suggest that the enzyme may be more active in the corpus region of the epididymis than in the caput and caudal regions.