The in vitro secretion of prolactin (PRL) and growth hormone (GH) by rat pituitary cells in primary monolayer cultures has been studied. The incorporation of 14C-l-leucine into these hormones was followed by subjecting the incubation medium to polyacrilamide gel electrophoresis. A dose of 0.5 micrograms/ml 17 beta-esatradiol increased the release of PRL and GH after 3 h of incubation. The basal release of PRL, but not of GH, was decreased 3 h after treatment with 0.5 micrograms/ml dopamine. Dopamine completely blocked the stimulatory effect of 17 beta-estradiol, but only partially the effect of Na-dibutyryl-3',5'-cyclic AMP (dbcAMP) on PRL secretion. The secretion of PRL and GH by pituitary cells after 3 days in culture from ovariectomized rats was lower than in cultures from intact rats and ovariectomized rats treated in vivo with estradiol benzoate (EB). These results suggest that depending on the estradiol levels in the blood the quantity of PRL and GH secreting cells in the anterior pituitary before preparation of the cell cultures may change. Furthermore, estradiol treatment in vivo can produce stable intracellular changes in the rate of synthesis and secretion of these hormones.