A study of the in vitro effect of dopamine on the release of newly synthesized prolactin is reported. Pituitary glands of female rats were incubated with 4,5-3H-leucine and the radioactive prolactin present in the pituitary gland and that released into the incubation medium were measured. Incubation with 5 × l 10–7M dopamine caused an 85% decrease in prolactin release. Prior injection of the rats with perphenazine or haloperidol rendered the pituitary gland refractory to the in vitro inhibitory effect of dopamine. Although in vitro perphenazine and haloperidol had little or no effect on release of prolactin, 5 × 10–9M of these drugs directly blocked the in vitro action of dopamine on prolactin release. Phentolamine, an α-blocking agent, was partially able to block the inhibitory effect, of dopamine. Propranolol, however, was not effective. Apomorphine, an agent known to mimic the effects of dopamine, caused a significant decrease in the amount of radioactive and radioimmunoassayable prolactin released into the incubation medium. This in vitro effect of apomorphine was blocked by the in vitro presence of perphenazine. Apomorphine did not block the effect of dopamine. 3 × 10–9M ergocryptine caused a complete inhibition of prolactin release but the effect of this drug, which binds to aα-adrenergic receptors, was completely blocked by the in vitro presence of perphenazine or haloperidol. The results of this study demonstrate that agents known to stimulate a-adrenergic or dopaminergic receptors cause an inhibition in prolactin release. Since hypothalamic catecholamines, including dopamine, have an important role in the control of prolactin secretion and because these catecholamines can directly inhibit the in vitro secretion of prolactin by the pituitary, consideration has been given to the hypothesis that dopamine is the physiological inhibitor of prolactin secretion that acts directly on the pituitary gland by stimulating specific receptors. (Endocrinology94: 1077, 1974)