Bovine granulosa cells were isolated from small antral, medium antral, and large Graffian follicles (i.e. small, medium, and large preovulatory follicles). Serum-free cultures of granulosa cells were established and found to be viable for 3-6 days of cell culture. Radiolabeled granulosa cell-secreted proteins were obtained and analyzed electrophoretically. No major changes were detected in the protein profiles of small, medium, and large follicle granulosa cells. FSH and insulin, however, had a dramatic effect on granulosa cell-secreted proteins and increased the apparent production of 200K, 65K, 25K, and 15K proteins. The effects of these hormones on the radiolabeled secreted proteins were similar for small, medium, and large follicle granulosa cells. Aromatase activity was high for the first day of serum-free granulosa cell culture and subsequently declined to low levels. Both FSH and insulin alone stimulated aromatase activity, while a combination of hormones resulted in an additive response similar to the stimulation observed with 10% calf serum. Although the level of aromatase activity increased slightly with the size of the follicle, the effects of hormones were independent of follicle size. Progesterone production was low on days 1 and 2 of serum-free granulosa cell culture and high on days 3 and 6 of cell culture. Interestingly, FSH and insulin suppressed progesterone production on day 1 of cell culture for small and medium follicle granulosa cells, but not for large follicle cells. In contrast, hormones stimulated progesterone production on days 3 and 6 of granulosa cell culture, and the level of progesterone production increased with the size of the follicle. The stimulatory effects of hormones on days 3 and 6 of the culture were similar for medium and large follicle granulosa cells, but were altered for small follicle cells. Results indicate that when aromatase activity is high and stimulated by hormones, progesterone levels are low and generally suppressed by the same regulatory agents. Conversely when progesterone levels are high and hormone responsive, aromatase activity is low. The inverse relationship between aromatase activity and progesterone production implies that bovine granulosa cells alter their differentiated state in culture from an estrogen-producing cell to a progesterone-producing cell. Combined observations indicate that the results obtained on day 1 of culture probably reflect the developmental and hormonal regulation of granulosa cell function in the preovulatory follicle, while data obtained at later times in culture reflect the ability of the cell to synthesize progesterone and develop a luteinization-like activity.(ABSTRACT TRUNCATED AT 400 WORDS)