The electrophoretic distribution of the polypeptides synthesized by bovine granulosa cell cultures after metabolic labeling with [35S]methionine has been analyzed by double gel electrophoresis. The fluctuations of 35 polypeptides have been followed as a function of the size of the follicles from which cultures originated, as a function of the cultures' proliferative stage (sparce, actively growing vs. confluent, resting cultures), and finally as a function of whether cells were exposed to either epidermal or fibroblast growth factor. When the patterns of protein synthesis in sparce vs. confluent granulosa cell cultures derived from small-sized follicles were compared, only a few differences were observed. In confluent cultures, 6 new peptides appeared, while 1 peptide present in sparce cultures disappeared. Cultures maintained in the presence of fibroblast or epidermal growth factor synthesized 20 new peptides upon reaching confluence. Among these were the 6 new peptides present in confluent but not in sparse granulosa cell cultures maintained in the absence of growth factors. The changes in protein synthesis observed in cultures grown in the presence of growth factors may reflect their direct effect on the cellular metabolism. A comparison between the protein distribution in cells derived from small- vs. large-sized follicles showed that fewer proteins were ultimately produced at confluence in cells derived from large-size follicles than in cells derived from small-sized follicles. This could be related to the process of cellular differentiation taking place within granulosa cells. The patterns of [35S]methionine-labeled proteins secreted by the granulosa cells into the incubation medium were analyzed and found to be similar regardless of the size of the follicle from which the culture originated. Little similarity between the proteins present in the follicular fluid and the pattern of the labeled proteins secreted into the incubation medium by cultured granulosa cells was observed. Only three proteins were identified which comigrate with proteins present in the follicular fluid. One of these was identified as fibronectin. This raises the possibility that the fibronectin present in the follicular fluid originated from granulosa cells and is not derived from plasma.