Progesterone (P4) regulation of FSH production was studied in dispersed ovine pituitary cell culture. FSH secretion into culture media was decreased by 30-70% in the presence of 10(-7) M P4. The ED50 of P4 was 2 X 10(-10) M. Decreased FSH secretion was observed as early as 9-12 h after treatment with P4 (10(-7) M). FSH secretion rebounded to control levels after P4 was removed for 4 days. The intracellular FSH content also decreased after P4 treatment, suggesting that decreased FSH secretion is due to decreased biosynthesis. Two progesterone metabolites, 5 alpha-dihydroprogesterone and 17 alpha-hydroxyprogesterone, showed relative activities of 39% and 28%, respectively, compared to P4. Although testosterone (10(-7) M) inhibited FSH secretion by 10%, it may have done so only via conversion to estradiol. Dihydrotestosterone, androstanedione, and cortisol had no effect on FSH secretion. P4 consistently showed activity in 2-day-old ovine culture, but its effect essentially disappeared by day 10 or 12 of culture. When insulin was included in the culture medium (medium 199 plus 10% charcoal-treated anestrous ewe serum), the effect of P4 was maintained for up to 20 days, and FSH production, in general, was augmented with insulin. These results indicate that P4 can specifically inhibit FSH production in ovine pituitary cell culture, but there is no long term maintenance of the P4 response unless insulin is present. Finally, it is known that 17 beta-estradiol also inhibits FSH production in ovine pituitary cultures. Therefore, our results establish that FSH production is a phenomenon that is regulated similarly by both estradiol and P4.