Serotonin (5-HT) stimulation of prolactin (PRL) secretion is mediated through the dopaminergic (DAergic) system, with 5-HT ligands having no direct effect on pituitary PRL release. Infusion of 5-HT into the third ventricle (ICV) or electrical stimulation (ES) of the medial preoptic area (POM) or the ventromedial nucleus (VMN) induces an increase in circulating PRL in the turkey. These increases in PRL do not occur when a selective antagonist blocks the D(1) dopamine (DA) receptors in the infundibular area (INF). In this study, the ICV infusion of (R)(-)-DOI hydrochloride (DOI), a selective 5-HT(2A) eceptor agonist, caused PRL to increase. Pretreatment with Ketanserin tartrate salt (KETAN), a selective 5-HT(2A) receptor antagonist, blocked DOI-induced PRL secretion, attesting to the specificity of the response. DOI-induced PRL secretion was prevented when the D(1) DA receptors in the INF were blocked by the D(1) DA receptor antagonist, R(+)-SCH-23390 hydrochloride microinjection, suggesting that the DAergic activation of the vasoactive intestinal peptide (VIP)/PRL system is mediated by a stimulatory 5-HT(2A) receptor subtype. The DOI-induced PRL increase did not occur when (+/-)-8-OH-DPAT (DPAT) was concurrently infused. DPAT is a 5-T(1A) receptor agonist which appears to mediate the inhibitory influence of 5-HT on PRL secretion. When DPAT was microinjected directly into the VMN, it blocked the PRL release affected by ES in the POM. These data suggested that when 5-HT(2A) receptors are activated, they influence the release of DA to the INF. When 5-HT(1A) receptors are stimulated, they somehow inhibit the PRL-releasing actions of 5-HT(2A) receptors. This inhibition could take place centrally, or it could occur postsynaptically at the pituitary level. It is known that D(2) DA receptors in the pituitary antagonize PRL-releasing effect of VIP. A release of DA to the pituitary, initiated by 5-HT(1A) receptors, could effectively inhibit PRL secretion.