Activating transcription factor-3 (ATF-3) is one member of a large family of leucine zipper transcription factors which bind to promoters responsive to cAMP and phorbol ester at the related cAMP (CRE) and phorbol ester response elements. We report here that ATF-3 is coexpressed with the neuropeptide precursor proenkephalin in human neuroblastoma SK-N-MC cells. Cotransfection experiments indicate that activation of proenkephalin gene expression by ATF-3 is dependent upon both the catalytic subunit of the cAMP-dependent protein kinase and the CRE-2 element. The CRE-2 element is essential for second messenger-inducible expression and is known to bind AP-1-like transcription factors. ATF-3 expressed in bacteria or from rabbit reticulocyte lysates binds to the proenkephalin CRE-2 element as a homodimer and as a heterodimer with Jun-D, another activator of proenkephalin transcription. ATF-3 stimulates binding of Jun-D to the proenkephalin CRE-2 element and acts synergistically with Jun-D to induce proenkephalin gene expression. Sequential immunoprecipitations of ATF-3 from SK-N-MC cells expressing proenkephalin indicate that ATF-3 is complexed with Jun-D in vivo and that both proteins are highly phosphorylated. Together, our results suggest that ATF-3 may play an important role in the regulation of gene expression by cAMP-dependent intracellular signaling pathways.