Previous studies have shown that the production of prolactin (PRL) is increased in human endometrial stromal cells cultured with medroxyprogesterone acetate (MPA) for 3-5 days. In the present study, we have investigated the effect of prolonged treatment of progestin and anti-progestin, RU 486, on the production and synthesis of PRL. Stromal cells were isolated from human endometrium obtained from non-pregnant women and cultured for 20-30 days in medium RPMI 1640 with 2% fetal calf serum or supplemented with MPA, RU 486, alone or in sequence. The PRL content in medium was measured by radioimmunoassay. The production rate was estimated from the PRL content in medium accumulated in 24 h. The PRL production rate was progressively increased in stromal cells continuously treated with MPA for 30 days (greater than 100-fold over the control value, i.e. 0-0.01 microgram/0.1 mg cell DNA/day). RU 486 alone had no effect on the production of PRL. However, the production of PRL was increased by MPA in stromal cells pretreated with RU 486 indicating that the effect of RU 486 is reversible. When stromal cells were treated with MPA and RU 486 sequentially, RU 486 stimulated the PRL production (approximately 2-fold over the MPA-treated cells) for 2-3 days and then reduced to basal levels over a 5-day period. The stimulatory and inhibitory effects of RU 486 on PRL production in stromal cells pretreated with progestin was also observed in the rate of synthesis of PRL estimated by incubating the stromal cells with [35S]methionine and immuno-isolating the [35S]PRL with anti-PRL.(ABSTRACT TRUNCATED AT 250 WORDS)