Anterior pituitary glands from normal untreated rats synthesize and release the hormone prolactin (Prl) during incubation under in vitro conditions. Addition of dopamine (DA) greatly inhibits the release of Prl and to a lesser extent reduces Prl synthesis. When pituitary glands are incubated in the presence of reserpine, there is a similar significant dose-related inhibition of Prl release. This effect persists even in the presence of a DA antagonist (haloperidol) and after the depletion of hypothalamic amines by in vivo treatment with reserpine. Reserpine in vitro also inhibits release of newly synthesized growth hormone from the pituitary glands of male rats; however, this is not observed when female rats are studied. We conclude that the direct effect of reserpine to inhibit Prl release is apparently independent of any interaction with catecholamine systems and is mediated by other, presently undefined mechanisms.