Estradiol is likely involved in stimulating developmental changes in the ability of the rat pituitary to secrete prolactin. To investigate the possibility that these changes involve proliferation of prolactin cells, estradiol effects on pituitary growth and prolactin synthesis were examined. Estradiol treatment of immature female rats stimulates increases in pituitary weight, [3H] thymidine incorporation, DNA content and prolactin synthesis. Treatment of rats with the DNA synthesis inhibitor, hydroxyurea, partially blocked the ability of estradiol to stimulate prolactin synthesis suggesting that at least part of the effect of estrogen is due to cell proliferation. These results suggest that estrogen-induced proliferation of prolactin cells is involved in the developmental processes of the pituitary.