Oocyte-secreted growth differentiation factor (GDF) 9 and bone morphogenetic protein (BMP) 15 are critical regulatory factors in female reproduction. Together, they promote granulosa cell proliferation and stimulate the maturation of preovulatory follicles. Despite their importance in female fertility, GDF9 and BMP15 expression patterns and function during spermatogenesis have not been investigated. In this study we show that the expression and stage-specific localization of both factors are limited to the germ cells of the rat seminiferous epithelium, with GDF9 being principally localized in round spermatids and BMP15 in gonocytes and pachytene spermatocytes. To identify potential cellular targets for GDF9 actions, cells of the seminiferous tubule were isolated and screened for the expression of signaling receptors [activin-like kinase (ALK) 5, ALK6, and BMP receptor, type II)]. Individual receptor types were expressed throughout the seminiferous epithelium, but coexpression of ALK5 and BMP receptor, type II was limited to Sertoli cells and round spermatids. Based on the reproductive actions of related TGFbeta ligands in the ovary and testis, GDF9 was assessed for its ability to regulate tight junction function and inhibin B production in rat Sertoli cell cultures. When recombinant mouse GDF9 was added to immature Sertoli cell cultures, it inhibited membrane localization of the junctional proteins claudin-11, occludin, and zonula occludens-1, thereby disrupting tight junction integrity. Concomitantly, GDF9 up-regulated inhibin subunit expression and significantly stimulated dimeric inhibin B protein production. Together, these results demonstrate that GDF9 and BMP15 are germ cell-specific factors in the rat testis, and that GDF9 can modulate key Sertoli cell functions.