Studies on the mechanisms which govern the release of prolactin were undertaken using two in vitro techniques. A dispersed preparation of rat anterior pituitary cells was made by mechanical means in the presence of trypsin. These washed cells were drawn up into a small column together with a Bio-Gel matrix and perifused with Earle’s basic salt solution. The eluates containing prolactin were then collected at short intervals. Test substances were added to the perifusion medium and their effect on prolactin release was measured. The results of these studies were compared with those obtained by incubating hemipituitary glands in Medium 199 to measure the effect of test substances on the release of radioimmunoassayable prolactin. Perifusion of dispersed pituitary cells with dopamine produced a marked inhibition of prolactin release within 3 min, and maximal suppression was noted 11 min after initiating the perifusion. Upon withdrawal of dopamine, prolactin release began to recover within 1 min and continued to rise to 80% of baseline at 6.5 min. Perifusion of pituitary cells in medium free of calcium also produced a marked reduction in prolactin release which was restored after reexposure of the cells to calcium. The addition of manganese and D-600, agents which block calcium channels, also caused reversible inhibition of prolactin release. The effects of the ionophores A23187 and X537A on prolactin release were studied. The presence of calcium ionophore A23187 did not effect prolactin release but it reversed the dopamine-mediated inhibition of prolactin release. In the absence of calcium, both ionophores stimulated release of prolactin. Tetrodotoxin, a blocker of sodium channels had no effect on prolactin release. Agents such as prostaglandin E1, and cholera toxin increased cyclic AMP levels, but no positive correlation was obtained on prolactin release patterns. Gpp(NH)p-stimulated adenylate cyclase activity in homogenates of anterior pituitary tissue was unaffected by dopamine. In contrast, addition of dibutyryl cyclic AMP to perifused pituitary glands stimulated prolactin release and theophylline added to hemipituitary gland completely reversed the inhibitory effect of dopamine on prolactin release and caused a concomitant increase in cyclic AMP levels. It is suggested that the tonic high level of prolactin release is maintained by influx of extracellular calcium and that dopamine inhibits this process. The role of intracellular cyclic AMP is undefined; however, the effects of dibutyryl cyclic AMP and theophylline may be due to mobilization of intracellular calcium and thereby stimulate prolactin release by this mechanism rather than through cyclic AMP. In summary, we present evidence that regulation of prolactin secretion by normal lactotropes is a calcium-mediated proces