Osteopontin (OPN), a multifunctional phosphoprotein found in both hard and soft tissues, was examined in the male reproductive tract. The expression and regulation of OPN in the rat testis, efferent ducts, and epididymis was examined during postnatal development through to adulthood using immunocytochemistry at the light- and electron-microscopic level. Immunoblot analysis revealed a major 30-kDa band for epididymal tissue and a major 60-kDa band for the testis. In the testis, immunostaining of OPN was noted in early germ cells from spermatogonia to early pachytene spermatocytes, suggesting a role for OPN as an adhesive protein binding these cells to the basement membrane and adjacent Sertoli cells. Nonciliated cells of the efferent ducts expressed OPN, whereas a cell- and region-specific distribution of OPN was observed in the epididymis. Reactivity of OPN in the apical region of the cell corresponded to labeling of microvilli, small endocytic vesicles, and endosomes, where OPN may serve to remove calcium from the epididymal lumen and, thus, prevent mineral accumulation and subsequent decrease in sperm fertility. Regulation and postnatal studies revealed that circulating androgens regulate OPN expression in principal cells of the epididymis only. Taken together, the data reveal cell- and region-specific expression and regulation of OPN in the epididymis.