The prodynorphin gene encodes the precursor molecule from which the dynorphin family of opioid peptides is generated. The gene is transcriptionally active in a wide variety of brain regions and endocrine tissues. Much is known regarding the physiological and receptor-mediated events that regulate prodynorphin gene expression in vivo. However, the molecular mechanisms by which specific cis- and trans-acting factors control activity of the prodynorphin promoter are not as clearly defined. In the study described here, transient transfection of prodynorphin promoter-chloramphenicol acetyl transferase plasmid constructs into CV1 cells served to identify three nucleotide sequence elements conforming to cAMP regulatory element motifs which regulate both basal and protein kinase A (PKA)-induced transcription. The three elements are clustered at positions -1543, -1627, and -1659 relative to the RNA cap site. Site-specific mutagenesis further reveals that although the sites can act independently to positively regulate transcription from the prodynorphin promoter, they can also act combinatorially to produce maximal transcriptional efficacy. Gel retention analysis employing rat brain protein extracts also describes the ability of these sequence elements to form sequence-specific DNA/protein complexes.