Transcription of the neurotransmitter biosynthetic genes tyrosine hydroxylase and dopamine beta-hydroxylase (DBH) is regulated by cell type-specific transcription factors, including the homeoprotein Arix, and second messengers, including cyclic AMP. The cis-acting regulatory sites of the DBH gene which respond to Arix and cAMP lie adjacent to each other, between bases -180 and -150, in a regulatory element named DB1. Neither Arix nor cyclic AMP analogs alone effectively stimulate transcription from the DBH promoter in non-neuronal cell cultures. However, when Arix is present together with cAMP, transcription is substantially activated. Synergistic transcription from the DBH promoter can also be elicited by cotransfection of Arix with an expression vector encoding the catalytic subunit of protein kinase A. Nuclear extracts from PC12 cells display a cAMP-induced complex binding to the DB1 element, and antisera to transcription factors CREB, CREM, Fos, and Jun indicate that these proteins, or closely related family members, interact with DB1. A dominant negative construct of CREB inhibits the response of the DBH promoter to protein kinase A. These results demonstrate a synergistic interaction between a homeodomain protein and the cAMP signal transduction system and suggest that similar interactions may regulate the tissue-specific expression of neuroendocrine genes.