Inhibin and activin are related proteins thought to be potential paracrine regulators of testicular development and maintenance of spermatogenesis. Messenger RNA and proteins immunologically related to both factors have been identified in the adult testis. However, the role(s) of these factors in paracrine regulation of testicular function is poorly understood. To identify potential targets for inhibin and activin in immature and adult testis, we used in situ binding of [125I]-labeled ligands to localize and describe the distribution of binding sites for inhibin and activin in testes of 15-, 18-, 21-, 30-, 45-, and 60-day-old rats. Nonspecific binding was defined as that occurring in the presence of a 1000-fold excess of unlabeled recombinant human (rh) inhibin or activin. [125I]-Inhibin was found to bind to interstitial cells throughout development. Inhibin binding was shown to co-localize with cells that showed positive staining for 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD). Competition studies demonstrated that this binding was indeed specific for inhibin. In contrast, [125I]-activin showed two distinct patterns of binding. First, [125I]-activin was shown to bind in a non-stage-dependent manner to cells located in the basal compartment of the seminiferous tubules in testis obtained from animals of all ages studied. Binding of [125I]-activin in the periphery of the tubule could be inhibited entirely by coincubation with excess unlabeled activin and partially with excess unlabeled inhibin. The ability of inhibin to compete with activin for binding appeared to be more pronounced in younger animals. In 45- and 60-day-old animals, a second stage-dependent component of [125I]-activin binding was also apparent. This binding was localized to spermatids found in stage VII-VIII tubules and was inhibited by the presence of excess activin, but not inhibin. These results indicate that inhibin can bind specifically to testicular interstitial cells throughout development and may be an important regulator of Leydig cell testosterone production or interstitial cell function. In contrast, activin appears to bind in a specific and stage-dependent manner to receptors or high-affinity binding proteins on spermatids as well as to sites on the periphery of all seminiferous tubules. These results support the hypothesis that both activin and inhibin may act at several levels to regulate proliferation or differentiation of germ and Sertoli cell function as well as to modulate interstitial cell activity.