H ypothalamic dopamine inhibits pituitary prolactin secretion and proliferation of prolactin-producing lactotroph cells by activating lactotroph dopamine D2 receptors (D2Rs). Conversely, prolactin (PRL) stimulates hypothalamic dopamine neurons via PRL receptors (PRLRs) in a short-loop feedback circuit. We used Drd2^–/– and Prlr^–/– mutant mice to bypass this feedback and investigate possible dopamine-independent effects of PRL on lactotroph function. The absence of either receptor induced hyperprolactinemia and large prolactinomas in females. Small macroadenomas developed in aged Prlr^–/– males, but only microscopic adenomas were found in Drd2^–/– male mice. Pharmacologic studies in Prlr^–/– mice with D2R agonists and antagonists demonstrated a significant loss of endogenous dopamine tone, i.e., constitutive inhibitory signaling by the D2R, in the pituitary. However, Prlr^–/– mice exhibited more profound hyperprolactinemia and larger tumors than did age-matched Drd2^–/– mice, and there were additive effects in compound homozygous mutant male mice. In vitro, PRL treatment markedly inhibited the proliferation of wild-type female and male Drd2^–/– lactotrophs, but had no effect on female Drd2^–/– lactotrophs, suggesting a downregulation or desensitization of PRLR in response to chronic hyperprolactinemia. We conclude that PRL inhibits lactotrophs by two distinct mechanisms: (a) indirectly by activation of hypothalamic dopamine neurons and (b) directly within the pituitary in a dopamine-independent fashion.