Previous in vitro and in vivo studies from our laboratory showed that progesterone (P₄), corticosterone (B), and testosterone (T) increase intracellular content and release of FSH in the anterior pituitary. Activin (Act) and inhibin (Inh) are structurally related proteins with antagonistic actions, as Act stimulates and Inh inhibits FSH secretion from the anterior pituitary. Together with follistatin (FS), a protein that bioneutralizes Act, they form an autocrine-paracrine loop in the anterior pituitary that tightly regulates FSH secretion. The objective of the present study was to test the hypothesis that P₄, B, and T modulate this autocrine-paracrine loop to favor increased FSH secretion. If Act were to mediate steroid-induced FSH release, FS would be expected to block these effects. To test this interaction, cell cultures were prepared from anterior pituitaries of male and female rats, and treated with Act, B, P₄, or T in the absence or presence of FS. Act, B, P₄, and T increased FSH release; FS suppressed both basal and Act- and steroid-stimulated FSH release to approximately 50% below basal levels. Cell cultures from anterior pituitary of female rats were used to compare the interaction of incremental concentrations of FS on dose-related Act- and P₄-stimulated FSH release. With increasing concentrations of Act, the FS-induced suppression of FSH release was attenuated and eventually abolished; in contrast, maximally stimulatory concentrations of P₄ did not fully overcome the FS-induced suppression of FSH release. The effects of P₄, B, and Act in the presence and absence of estradiol on steady-state mRNA levels of FSHβ, Actβ_B, and FS were determined in primary pituitary cell cultures from metestrous female rats by reverse transcription-polymerase chain reaction. Whereas Act, P₄, B increased FSHβ mRNA levels, only Act raised the level of FS mRNA, and neither steroid increased Actβ_B mRNA. The results support the hypothesis that endogenous Act is a common mediator of the action of P₄, B, and T in the rat primary anterior pituitary cell culture. We conclude that the stimulation of FSH release and intracellular content in the gonadotroph by P₄, B, and T is mediated, in part, by Act and involves modulation of a tightly regulated Act/FS autocrine-paracrine loop.