The intraovarian function of gonadally produced activin is unclear, and many in vitro studies have suggested a role for activin in follicle development. To identify the follicular developmental stages at which these ligands may be acting, we have used immunohistochemical localization of the ligand subunits, receptor subtypes, and Smad co-activating proteins within the same follicles. The earliest stages of follicle development (primordial to primary) show no immunoreactivity for the activin subunits or their receptors. Oocytes from these early stages contain immunostaining for Smad2 and Smad4, consistent with signaling by other TGF-beta superfamily members. Immunostaining for the activin type II receptor first appears in oocytes and oocyte-associated cumulus cells at the secondary follicle stage. However, activin is not produced in these follicles, suggesting that either the receptors are inactive at this stage or they are used by another protein. Co-localization of activin and inhibin subunits, receptors, and Smads only occurs in granulosa and theca cells of small antral, aromatase-positive follicles as well as granulosa cells of early atretic follicles. In addition, multivariate statistical analysis reveals that the ligands and their cellular signaling complexes are independently regulated. Together, these data strongly suggest that the intraovarian role of activin is limited to a few developmental stages and that other TGF-beta family members may use this cell autonomous signaling machinery in early follicle development.