In this present study, we investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor, PACAP type I receptor (PAC1R) on prolactin synthesis in pituitary somatolactotroph GH3 cells. PACAP increased prolactin promoter activity up to 1.3 ± 0.1-fold. This increase, while significant, was less than the increase resulting from thyrotropin-releasing hormone (TRH) stimulation. By transfection of a PAC1R expression vector to the cells, the response to PACAP on prolactin promoter activity was dramatically potentiated to a degree proportional to the amount of PAC1R transfected. In the PAC1R expressing GH3 cells, TRH and PACAP alone increased prolactin promoter up to 3.3 ± 0.3-fold and 4.9 ± 0.2-fold, respectively, and combined treatment with TRH and PACAP further increased prolactin promoters up to 6.8 ± 0.6-fold. PACAP binds both Gs- and Gq-coupled receptors and stimulates adenylate cyclase/cAMP and protein kinase C/extracellular signal-regulated kinase (ERK) signaling pathways. PACAP increased ERK phosphorylation in PAC1R expressing cells to the same degree as TRH. Combined treatment with TRH and PACAP had a synergistic effect on ERK activation. GH3 cells produce both prolactin and growth hormone. Stimulation of GH3 cells with TRH significantly increased the mRNA level of prolactin and attenuated growth hormone mRNA expression. PACAP increased both prolactin and growth hormone mRNA levels, particularly in PAC1R expressing cells. In addition, increasing amount of PAC1R in GH3 cells potentiated the action of TRH on prolactin promoter activity, as well as on ERK phosphorylation. PAC1R was induced by PACAP itself, but not by TRH. Our current study demonstrates that PACAP and its PAC1R, functions as a stimulator of prolactin alone or with TRH in prolactin producing cells.