We have found that exogenous prolactin (PRL) stimulates all three populations of hypothalamic neuroendocrine dopaminergic neurons. In this study, we investigated the effects of immunoneutralization of endogenous PRL on the activity of these neurons. Injection of 17beta-estradiol (E2) (20 microg subcutaneously) 10 d after ovariectomy induced a proestrus-like increase in PRL in peripheral plasma the following afternoon. At 1000 h the day after E2 injection, rats received either rabbit antirat PRL antiserum (PRL-AS) (200 microL) or normal rabbit serum (NRS, 200 microL, controls) intraperitoneally. Groups of rats were then decapitated every 2 h from 1100 h to 2100 h. Trunk blood was collected and serum extracted with protein A to remove the PRL-AS/PRL complex, and the remaining free PRL was measured by radioimmunoassay. Sites of neuroendocrine dopaminergic nerve terminals, the median eminence (ME), and intermediate and neural lobes of the pituitary gland were excised and stored for determination of dopamine (DA) and 3,4-dihydroxyphenyl acetic acid (DOPAC) concentrations by high-performance liquid chromatography electrochemical detection (EC). In addition, the anterior lobe of the pituitary gland, the locus of DA action, was collected. The concentration of PRL in NRS-treated animals increased by 1500 h, peaked by 1700 h, and returned to low levels by 2100 h. PRL-AS prevented the increase in PRL secretion in response to E2. The turnover of DA (DOPAC:DA ratio; an index of dopaminergic neuronal activity) in the ME of NRS-treated animals increased at 1500 h and rapidly returned to basal levels. Treatment with PRL-AS prevented the increase in DA turnover in the ME. DA turnover in the intermediate lobe increased coincident with the peak of PRL in serum of NRS-treated rats. PRL-AS administration prevented increased DA turnover in the intermediate lobe. The turnover of DA in the neural lobe increased by 1300 h and decreased steadily through 2100 h. However, administration of PRL-AS minimally suppressed the turnover of DA in the neural lobe. Moreover, administration of PRL-AS attenuated the rise of DA in the anterior lobe associated with the waning phase of the E2-induced PRL surge. These results clearly indicate that endogenous PRL regulates its own secretion by activating hypothalamic neuroendocrine dopaminergic neurons.