Constitutively active mutations of the G protein alpha(S) subunit are detected at a high frequency in human pituitary adenomas that secrete GH or PRL. It seems possible that over-expression of the pituitary cell-specific transcription factor Pit-1/GHF-1 (Pit-1) gene in response to active alpha(S) subunits contributes to the formation of these adenomas. We have examined whether expression in pituitary cells of one of these constitutively active alpha(S) subunits, Q227L-alpha(S), stimulates expression directed by the Pit-1 promoter. Transient expression of Q227L-alpha(S) yielded a strong stimulation of a target Pit-1 promoter-chloramphenicol acetyl transferase (CAT) construct, (-200)Pit-1-CAT. Expression of wild-type alpha(S) or an inactive alpha(S) mutant yielded, respectively, reduced or no stimulation of CAT activity. A dominant inhibitor of protein kinase A (PKA), RAB, blocked almost completely either forskolin (FSK) or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that PKA is required for the action of Q227L-alpha(S) on the Pit-1 promoter. The Pit-1 promoter contains a binding site for Pit-1 and two CREB binding sites. Mutation of the Pit-1 binding site reduced but did not eliminate either FSK or Q227L-alpha(S) stimulation of Pit-1 promoter activity, implying a partial but incomplete requirement for this element for a PKA-mediated response to Q227L-alpha(S). The CREB dominant inhibitor S133A-CREB yielded a partial reduction in either FSK or Q227L-alpha(S) stimulation of (-200)Pit-1-CAT expression, implying that one or both of the Pit-1 promoter adenosine 3'5'-monophosphate response element binding protein (CREB) binding sites is/are also required for a complete PKA-mediated response to Q227L-alpha(S). The observation that S133A-CREB completely blocked the response to FSK or Q227L-alpha(S) of a Pit-1 promoter containing a mutated site PitB1 implies that the binding sites for Pit-1 and CREB account for all of the response elements for FSK or alpha(S) in the Pit-1 promoter.