Mammalian sperm surface antigens are acquired either during spermatogenesis or sperm maturation in the epididymis. These antigens, many of which are hydrolytic enzymes, are actively synthesized and secreted by the resident epithelial cells and adsorbed to the sperm membrane as part of posttesticular sperm modification. In this study, we aimed to investigate the expression of cathepsin-D (CAT-D) in human reproductive tissues and its distribution on the sperm surface in different sperm conditions. Immunohistochemical results revealed the expression of CAT-D in the somatic Sertoli and Leydig cells without showing any immunoreactivity in any germ cells, despite their engagement of the acrosomal system. A strong immunoreactivity of anti-CAT-D was also detected in the epididymal epithelium, chiefly in the principal cells, which are known to actively synthesize and secrete proteins into the epididymal lumen. The absence of CAT-D in the clear cells was unexpected because these cells are known to engage the endosomal machinery. We further showed that CAT-D was anchored on the sperm surface confined to the postacrosomal region without any lateral redistribution within the membrane during sperm capacitation. However, the enzyme underwent changes to be an active form of a 29/30-kd doublet during sperm capacitation. Using CAT-D as a marker, we were able to demonstrate here localization of the enzyme in human reproductive tissues, as well as reveal membrane modification in human sperm during maturation and capacitation processes.