The release of trophic hormones by the anterior pituitary is regulated by hypothalamic factors but additionally can be influenced by factors produced and released within the anterior pituitary itself. Our aim was tostudy the possible presence of all essential elements of the serotoninergic system within the anterior pituitary and to characterize the effect5-HT might have on the hormone release. We found in the anterior pituitary, through RT-PCR analysis, the gene expression of the rate limiting enzyme necessary for the production of 5-HT, tryptophan hydroxylase, as well as the membranal serotonin transporter necessary for uptake and serotonin receptors (5HTR), with the expression of 5-HTR4, 5A, 5B, 6 + 2C under hormonal regulation during the culturing of anterior pituitary aggregates. Subsequent single cell RT-PCR on redispersed anterior pituitary cultured cells showed that the majority (62%) of cells expressing the mRNA for the peripheral form of tryptophan hydroxylase (TPH1) did not co-express the mRNA of an anterior pituitary hormone. Additionally, western blot analysis showed the presence of tryptophan hydroxylase protein in anterior pituitary aggregates, while immunofluorescence identified the presence of the serotonin transporter on GH cells. The effect of serotonin on hormone secretion was studied using the perifusion system, which allows for the continual observation of hormone secretion over time and during the application of various compounds. We found that nanomolar concentrations of 5-HT caused a dose dependant release of both Growth Hormone (GH) and Prolactin (PRL); however the presence of estradiol was necessaryin the culturing in order to observe the effect on PRL release. The PRLresponse to 5-HT was not inhibited by the non-selective 5-HTR antagonists methysergide and methiothepin nor by selective 5-HTR1, 5-HTR2, 5-HTR3, 5-HTR6 and 5-HTR7/5 antagonists, while, among different agonist analogues, only α-methyl-5-HT evoked a substantial PRL release. The 5-HTR4 antagonist GR113808 completely abolished the PRL response while the 5-HTR4 agonist cisapride stimulated PRL secretion. 5-HT doubled cAMP production in hormone-free-cultured aggregates and caused a 5-fold increase in cAMP in E2-treated aggregates. The effect of 5-HT on PRL release was not affected by blocking 5-HT uptake via the serotonin transporter or thevesicular monoamine transporter. The GH response to 5-HT was partially blocked by the non-selective 5-HTR antagonists methiothepin and methysergide (1 µM), the selective 5-HTR2B antagonist SB-206553 (1 µM) and the 5-HTR5/7 antagonist SB-269970 (1 µM). The 5-HTR1/ 5/ 7 agonist 5-CT, the 5-HTR7 agonist tryptamine and the 5-HTR2 agonists DOI, mCpp and α-methyl 5-HT all increased GH release. Gene expression analysis revealed no detectable expression of the 5-HTR5A, 5-HTR5B in aggregated cells cultured with no hormone supplement yet were detected in E2-treated aggregates. In aggregates cultured with no hormone supplement the response to 5-HT was inhibited in the presence of 5-HTR7/5 antagonist SB-269970, was less diminished than in E2-treated aggregates, and the 5-HTR2B antagonistSB-206553 inhibited the GH response to 5-HT almost completely. This implies that the 5-HTR7 is involved in the GH response to 5-HT but can not ruling out the involvement of the 5-HTR5 in E2-treated aggregates.We investigated whether 5-HT is present in anterior pituitary cultures through Reverse-Phase HPLC followed by mass spectrometry and found no detectable levels of 5-HT. Similarly we could not detect the production of 5-HT by anterior pituitary cells by radiometric or biochemical assays. We demonstrated that the anterior pituitary cells are capable of producing other endogenous ligands such as tryptamine, and provide circumstantial evidence for the production of 5-hydroxykynuramine.We can conclude that a serotoninergic system is present in the anteriorpituitary and that the 5-HTR4, is responsible for the increases in PRL,while the 5-HTR2B and 5-HTR7 are responsible for the increases in GH release. There is therefore a functional role for 5-HT as a modulator of GH and PRL secretion at the level of the pituitary.