Progestins stimulate prolactin production from endometrial stromal cells in culture. We compared the potencies of the synthetic progestins norethindrone and medroxyprogesterone acetate to natural progesterone in inducing stromal prolactin production. Modifications of the culture system provided an increase in stromal cell yield, thus permitting multiple comparisons from the treatment of cells from a common endometrial sample. The effects of high-dose estradiol also were evaluated in this system. The findings suggest relative potencies of 50:1 for medroxyprogesterone acetate and progesterone. Norethindrone gave intermediate and more variable responses. Estradiol potentiated prolactin production from only submaximal progestins doses. The differences between the progestin effects, in large measure, were due to differential culture growth as reflected by culture mass. Compared to controls and estradiol alone, all the progestins induced much greater prolactin production. Thus during decidualization, progestins probably promote both stromal growth and intracellular prolactin production. High-dose estradiol may not interfere with these events.