Expression of the gene encoding the neurotransmitter biosynthetic enzyme dopamine beta-hydroxylase (DBH) is regulated in a tissue-specific pattern, and transcription is influenced by environmental stimuli. Using the promoter proximal region of the rat DBH gene and nuclear extracts from SHSY-5Y neuroblastoma cells, a DNA-protein complex was identified that is competitive with oligonucleotides containing the recognition site of transcription factor AP-2. DNase footprint analysis identified an AP-2 binding site between -136 and -115 of the DBH promoter. Mutation of that AP-2 site results in a sevenfold reduction of basal reporter gene expression, but second messenger-stimulated activity is retained. Cotransfection of an AP-2 expression vector and a DBH promoter-reporter construct into cultured cells results in a sixfold stimulation of reporter gene expression, demonstrating the ability of AP-2 to trans-activate the DBH promoter. These results identify a new regulatory element on the rat DBH gene and suggest that the AP-2 site plays a role in maintaining basal levels of DBH transcription.