Pituitary prolactin biosynthesis is negatively regulated by hypothalamic dopamine through D₂ receptors in pituitary lactotrophs, but little is known about the direct effect of dopamine on gonadotrophs. In this study, the clonal gonadotroph-derived cell line, αT3-1, was used to examine whether gene expression of the pituitary gonadotropin α subunit, stimulated with GnRH or pituitary adenylate cyclase-activating polypeptide (PACAP), was controlled by dopamine D₂ receptor. Western blotting and reverse transcription-polymerase chain reaction analysis demonstrated the presence of dopamine D₂ receptors in αT3-1 cells. Both GnRH and PACAP increased α subunit gene expression. GnRH-induced α subunit gene expression was not affected by quinpirol, a specific dopamine D₂ receptor agonist. In contrast, PACAP-induced gene expression was significantly lower in the presence of quinpirol. The roles of extracellular signal-regulated kinase (ERK) and cAMP in the expression of the α subunit gene were examined. GnRH activated ERK, but PACAP did not, and the activation was not inhibited by quinpirol. GnRH-induced α subunit gene expression was completely inhibited by an ERK inhibitor, PD098059. Cyclic AMP accumulation in αT3-1 cells was increased by treatment with PACAP, and quinpirol inhibited this effect. GnRH did not affect cAMP production in these cells. These results suggest that in αT3-1 cells, dopamine D₂ receptors negatively regulate pituitary α subunit gene expression in association with the cAMP-dependent pathway, but not with the ERK pathway.