The responsiveness of granulosa cells to the gonadotropins and cAMP increases as ovarian follicles mature. To determine if this change in response might be related to either the content or cAMP-dependent phosphorylation of specific proteins, we labeled proteins in 30,000 X g supernatant fractions (cytosol) with [gamma-32P] ATP in the presence or absence of cAMP. Using two-dimensional gel electrophoresis, we observed that granulosa cells of preantral follicles exhibited low amounts of cAMP-dependent phosphorylation of two proteins with apparent molecular weights of 54,000-56,000 and 43,000. Using [32P]8-N3cAMP and photoaffinity labeling procedures, the Mr = 54,000-56,000 protein was identified as RII, the regulatory subunit of type II protein kinase. Polychromatic silver staining, as well as the photoaffinity labeling, revealed that RII exists in three forms, each of which was also labeled by [gamma-32P] ATP. Based on the relative isoelectric points and specific silver staining of highly purified actin and phosphorylated actin, the Mr = 43,000 protein has been provisionally identified as actin. Five proteins (Mr = 37,500, 27,500, 22,500, 19,000, and 15,000), in addition to RII and actin, were phosphorylated in cytosol of granulosa cells from preovulatory follicles. By adding increasing concentrations of exogenous catalytic subunit to the cytosols, we demonstrated that the content, as well as the phosphorylation of these proteins, was increased selectively in granulosa cells of antral follicles. By using hypophysectomized rats, we demonstrated that these five proteins are induced by follitropin (FSH). Because they were not present in cytosols of thecal cells or corpora lutea, they appear to be specific markers for granulosa cells. The content and phosphorylation of RII was also dramatically increased in cytosols of granulosa cells from antral follicles, whereas that of actin remained unchanged. These observations indicate that granulosa cell differentiation involves regulation by FSH of specific proteins which are substrates for cAMP-dependent protein kinase. Thus, FSH and cAMP appear to regulate the intracellular content and phosphorylation of a cAMP response system in granulosa cells. The extent to which RII and the five specific phosphoproteins themselves regulate granulosa cell responsiveness remains to be determined.