The effect of porcine follicular fluid on estradiol and progesterone secretion was examined using a rat granulosa cell culture with FSH and testosterone in the medium. Follicular fluids from small (less than 5 mm) and large (greater than 6 mm) follicles (SFFI, LFF1) were treated with charcoal, and then fractionated by filtration through an Amicon XM-50 and an PM-10 membrane. The addition of 25% SFF1 and LFF1 into the culture system significantly inhibited estradiol and progesterone secretion (P less than 0.005). These inhibitory activities were observed in PM-10 retentates (10,000-50,000 MW) and filtrates (less than 10,000 MW) of SFF1 and LFF1. The addition of XM-50 filtrates (less than 50,000 MW) of SFF1 and LFF1 caused a dose-dependent inhibition of estradiol and progesterone secretion. The dose-response relationship between the filtrates and estradiol secretion was linear with a significant correlation coefficient. The addition of the filtrates exerted no inhibitory effect on the growth of the cells cultured. XM-50 filtrate of LFF1 from a batch with a low ratio of small/large follicles showed a lower inhibitory activity on estradiol secretion than that of LFF1, while the inhibitory activities in both filtrates on progesterone secretion were almost equivalent. These results suggest that the follicular fluid of small porcine follicle contains nonsteroidal regulators capable of inhibiting estradiol and progesterone secretion by cultured rat granulosa cells, and that the estradiol secretion inhibitor activity decreases in the fluid of large follicle while the progesterone secretion inhibitor activity does not decrease in it.