Prolactin secretion from the anterior pituitary is tightly regulated by feedback onto the hypothalamic neuroendocrine dopaminergic (NEDA) neurons. Prolactin stimulates these neurons to synthesize and secrete dopamine, which acts via the pituitary portal vasculature to inhibit prolactin secretion from the pituitary lactotrophs. Despite the physiological importance of this feedback, relatively little is known about the signaling mechanisms responsible for prolactin-activation of NEDA neurons. This issue has been examined here using a cell culture preparation of the fetal rat mediobasal hypothalamus. Prolactin stimulated a time- and concentration-dependent increase in catecholamine synthesis, which was maximal after 60-120 min (1 μg/ml prolactin) and inhibited by the prolactin antagonist Δ1-9-G129R-hPRL. This prolactin response was accompanied by a rise in the site-specific (ser-19, -31, and -40) phosphorylation of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Consistent with this observation, the prolactin-induced increase in cateoholamine synthesis was abolished by inhibitors of protein kinase A and protein kinase C (PKC). Prolactin incubation also resulted in a PKC-dependent activation of the MAPK pathway, although this was not required for the stimulation of catecholamine synthesis. In addition to increasing TH phosphorylation and catecholamine synthesis, prolactin also increased TH mRNA expression. In contrast to catecholamine synthesis, this latter response was not suppressed by inhibition of protein kinase A or PKC. These results indicate that although prolactin controls catecholamine synthesis in NEDA neurons by regulating both TH activity and TH mRNA expression, it employs distinct, nonoverlapping, signaling pathways to achieve these ends.