The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle-stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8-fold within a 2-day period. In serum-free medium, human luteinized granulosa cells were able to incorporate 3H-thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, 3H-thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH- and estradiol-treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze-thawing, was stable for several weeks at -20 degrees C, became unstable at 4 degrees C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight > 30,000 dalton.(ABSTRACT TRUNCATED AT 250 WORDS)