We have previously demonstrated that 5-HT stimulates not only basal but also thyrotropin-releasing-hormone (TRH)-induced prolactin (PRL) release by acting directly at the pituitary gland level. In the present report, the participation of an autoparacrine action of VIP in the stimulatory effects of 5-HT and the involvement of the 5-HT<sub>2</sub> receptor type in mediating serotonin-induced PRL release have been examined. Cultured anterior pituitary cells from ovariectomized adult rats were incubated for 1 h in 1 ml of T<sub>3</sub>-supplemented medium with or without the test substances. The results obtained in the presence of T<sub>3</sub> confirm our previous observations, since treatment of the cells with 5-HT caused dose-dependent increases in basal PRL release, with an approximate EC<sub>50</sub> of 3.68 × 10<sup>–8</sup> M, and led to a significant potentiation (1.3-fold) of the TRH-induced PRL release. In order to evaluate the possible participation of vasoactive intestinal peptide (VIP) as mediator of the effects of 5-HT on PRL release, cells were incubated in the presence of 5-HT alone (3–1,000 n M) or 100 n M 5-HT plus 30 n M TRH, with or without 200 n M VIP antagonist (VIP-At): [D,4-Cl-Ph<sup>6</sup>,Leu<sup>17</sup>]VIP. VIP-At partially inhibited the release of PRL induced by 5-HT, both basal and TRHstimulated release. The stimulatory effect of 5-HT, however, was not eliminated by VIP-At, since the PRL released in response to 5-HT was still over the respective control ones. These results further support the findings suggesting that 5-HT acts directly at pituitary level by stimulating PRL release. Addition of the 5-HT<sub>2</sub> receptor antagonist, ketanserin (1 µ M) into the incubation medium resulted in the loss of cellular responsiveness to 5-HT, preventing not only the stimulatory effect of 5-HT on the basal but also on the TRH-induced PRL release. In conclusion, the results further strengthen the possibility that 5-HT increases the basal PRL release and potentiates the stimulatory effect of TRH by acting directly at the level of the lactotropes. These effects are not simply a consequence of autoparacrine action of VIP. In addition, it was shown that ketanserin completely antagonizes PRL response to 5-HT, indicating the involvement of the 5-HT<sub>2</sub> receptor type in mediating PRL release.