A relationship between prolactinin synthesis and release was studied using a dispersed adenohypophysial cell primary-culture system. Newly synthesized prolactin was identified by the amount of prolactin-associated tritium activity. No new prolactin was synthesized during a 1-h incubation with 3H-Leu. After 4 h a significant amount of newly synthesized prolact was released into the medium but no labelled prolactin was detected in the cells. Radioimmunoassayable prolactin, however, was equally divided between the cells and the medium indicating that the newly synthesized prolactin was preferentially secreted. In contrast, approx. 2 times more newly labelled GH was found in the cell extract than in the medium while by GH-radioimmunoassay, the cells contained twice as much GH as the medium in the control group. During the 1-h incubation, estradiol (1.0 ng/ml) increased prolactin content in the medium, although no newly synthesized prolactin appeared in the medium or the cells. During the 4-h incubation period significantly more labelled prolactin was released into the medium in the presence of estradiol (1.0 ng/ml) than in the control. These results indicate that (1) estradiol has a stimulatory effect on prolactin secretion, (2) estradiol activates a prolactin-synthesis mechanism, (3) newly synthesized prolactin is preferentially released, and (4) the rate of release of newly synthesized prolactin and GH having different mechanisms of hypothalamic control, an inhibiting factor for the prolactin and a releasing factor for GH.