Hormone regulation of anterior pituitary expression of the common glycoprotein hormone -subunit ( GSU) is mediated by multiple response elements residing in the first -435 bp of the human promoter. In rat pituitary cells and mouse T3-1 precursor gonadotrophs, the human GSU promoter is strongly responsive to activators of the adenylyl cyclase/cAMP pathway, such as the hypothalamic releasing hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) and forskolin (an adenylyl cyclase activator). However, the role of PACAP and cAMP in regulating GSU transcription in the more differentiated L T2 gonadotroph is unclear. Here, we investigate the regulation of the human GSU promoter by PACAP and forskolin in L T2 and T3-1 gonadotrophs. PACAP failed to stimulate GSU promoter activity or cAMP production in L T2 cells, in marked contrast to T3-1 cells. L T2 gonadotrophs expressed extremely low levels of any PACAP type 1 receptors (PAC 1 -R) isoform by RT-PCR and lacked PAC 1 -R by radioligand binding. Forskolin stimulated the GSU promoter in L T2 cells, but by less than 30% of the response seen in T3-1 gonadotrophs. This blunted cAMP transcriptional effect was not due to different levels of cAMP generation, or altered expression of the cAMP target proteins CREB, Akt, CBP or ICER. However, only L T2 cells showed detectable expression of the protein kinase A type II regulatory subunit. Binding of activating transcription factor-2 and phosphorylated CREB to the consensus CRE was observed in both L T2 and T3-1 gonadotrophs, yet forskolin failed to stimulate either CRE-or CREB-mediated transcription in L T2 cells. Collectively, these data demonstrate the lack of functional PACAP receptors in L T2 gonadotrophs, and a pronounced attenuation in the responsiveness of this differentiated gonadotroph cell line to cAMP stimulus.