Pituitary cells cultured with estradiol respond by a specific increase in prolactin synthesis. Extensive inhibition of DNA synthesis (61-78%) with hydroxyurea or cytosine arabinoside resulted in only 28-33% decrease in estrogen-induced prolactin synthesis. To assess the role of prolactin cell proliferation in the estrogen-induced response, mammotrophs were identified by immunocytochemistry. Cultures treated with estradiol for 1, 2 or 5 days contained 101 +/- 1, 113 +/- 2 and 132 +/- 1% of the number of mammotrophs in controls. Estradiol treatment for corresponding periods resulted in prolactin synthesis representing 94 +/- 5, 144 +/- 11 and 270 +/- 22% of controls and prolactin mRNA levels representing 115 +/- 7, 160 +/- 7 and 322 +/- 22% of controls. Thus estrogen caused a considerable increase in prolactin synthesis which paralleled the increase in prolactin mRNA levels and a much smaller relative increase in the number of mammotrophs. We conclude that regulation of prolactin synthesis by estrogen is mediated predominantly but not exclusively through stimulation of gene expression in existing pituitary cells.