The time course of inhibition by gonadotropinreleasing hormone (GnRH) of granulosa cell differentiation was analyzed in follicular cells cultured from ovaries of hypophysectomized diethylstilbestrol-implanted rats. Addition of FSH caused a 150-fold rise in cAMP production during the first 30 min of granulosa cell culture, followed by a plateau until 30 h and a secondary increase from 30 to 48 h. The GnRH analog, [D-Ala6]des-Gly10-GnRH iV-ethylamide (GnRHa), transiently inhibited cAMP production during the first 30 min, but from 1–24 h of culture, cAMP levels were similar in FSH-and FSH plus GnRHa-treated cells. Thereafter, GnRHa caused a marked fall in cAMP levels from 30–48 h of culture. The phosphodiesterase inhibitor, l-methyl-3-isobutylxanthine, completely abolished the inhibitory effect of GnRHa on cAMP accumulation. Addition of GnRHa with FSH or at times up to 24 h of culture suppressed FSH-induced cAMP accumulation, LH receptor formation, and progesterone production at 48 h. Later addition of GnRHa, at 30 or 42 h of culture, prevented the progressive development of FSH-induced responses, which remained at the preexisting levels. The inhibitory actions of GnRHa upon granulosa cell function appeared to be irreversible, since cells cultured with GnRHa from 0–4 h or from 24–30 h showed no further accumulation of cAMP and progesterone at 48 h. The morphological changes induced by FSH were not affected by GnRHa during the first 24 h of culture, with comparable formation of multilayered aggregates of epithelial-like cells. However, from 24–48 h, FSH maintained cellular aggregation and the epithelial appearance of the cells, while GnRHa caused regression of cellular aggregation with marked flattening into monolayers. These results indicate that the presence of GnRHa is necessary for only a few hours to inhibit FSH action in cultured granulosa cells, and that the hypothalamic peptide analog causes cessation of FSH action when added at any time during the differentiation process.