Arylsulfatase A (AS-A) is localized to the sperm surface and participates in sperm-zona pellucida binding. We investigated how AS-A, usually known as an acrosomal enzyme, trafficked to the sperm surface. Immunocytochemistry of the mouse testis confirmed the existence of AS-A in the acrosomal region of round and elongating spermatids. However, immunofluorescence and flow cytometry indicated the absence of AS-A on the surface of live testicular sperm. In contrast, positive AS-A staining was observed in the heads of live caudal epididymal and vas deferens sperm. The results suggested that acquisition of AS-A on the sperm surface occurred during epididymal transit. Immunocytochemistry of the epididymis revealed AS-A in narrow and apical cells in the initial segment and in clear cells in all epididymal regions. However, these epithelial cells are in the minority and are not involved in secretory activity. In the caudal epididymis and vas deferens, AS-A was also localized to principal cells, the major epithelial cells. Because principal cells have secretory activity, they may secrete AS-A into the epididymal fluid. This hypothesis was supported by our results revealing the presence of AS-A in the epididymal and vas deferens fluid (determined by immunoblotting and ELISA) and an AS-A transcript in the epididymis (by reverse transcription polymerase chain reaction). Alexa-430 AS-A bound to epididymal sperm with high affinity (K_d = 46 nM). This binding was inhibited by treatment of sperm with an antibody against sperm surface sulfogalactosylglycerolipid. This finding suggests that AS-A in the epididymal fluid may deposit onto sperm via its affinity to sulfogalactosylglycerolipid.