Prodynorphin transcription is activated via Gs-coupled receptors through a cyclic AMP (cAMP)-dependent pathway. Four cAMP response elements (CREs) are present within the rat prodynorphin (RD) control region, and all four CREs appear to function in RD regulation. Three CREs located upstream between -1860 and -1504 are critical for receptor-responsive activity, but their function is distance dependent unless they act together with a fourth CRE found in exon 1. Regulation of RD also appears to involve multiple CRE-binding proteins. Both CRE-binding protein (CREB) and activator protein 1 (AP-1) can regulate RD, but their effects are in opposite directions; CREB represses and AP-1 activates RD. CREB-induced repression and AP-1 activation require distinct elements within the control region, but their binding and functions overlap at CRE-3. While CREB repression is dependent on CRE-3, AP-1 activation (and cAMP induction) of RD requires additional CREs (CRE-1, -2, and -4). CREB repression blocks AP-1 activation in unstimulated cells. However, phosphorylation relieves CREB-induced repression and enhances AP-1 activation. Gs-coupled receptor activation of RD may require phosphorylation-dependent derepression and activation steps.