It has been reported that the highly homologous neuropeptides pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal polypeptide (VIP) exert similar cardiovascular effects in vivo. In the present study we compared the effects of these neuropeptides on myocardial cyclic AMP content and the release of immunoreactive CDD/ANP-(99–126) (atrial natriuretic peptide). In cultured neonatal rat cardiomyocytes PACAP and VIP evoke concentration-dependent increases in intracellular cyclic AMP content but responses to VIP are markedly less. PACAP stimulates the release of CDD/ANP-(99–126) in a concentration-dependent manner with a threshold concentration of 1 nM, and up to a 6-fold increase in basal secretion at 1 μM PACAP. In contrast, VIP had no effect on the release of CDD/ANP. Pretreatment of cells with the competitive PACAP-antagonist, PACAP-6-38 (1 μM), significantly reduces the effects of PACAP on intracellular cyclic AMP and on CDD/ANP-(99–126) secretion and abolishes the effects of VIP on cyclic AMP. Pretreatment with VIP-receptor antagonist (1 μM) prevents the cyclic AMP-responses to VIP while increases in cyclic AMP as well as stimulation of CDD/ANP-(99–126) release by PACAP are not affected. It is concluded that both neuropeptides directly influence cardiac myocytes through an increase in intracellular cyclic AMP. Release of CDD/ANP-(99–126) by PACAP may be involved in the decrease in blood pressure that follows intravenous administration of this peptide. The higher potency of PACAP to induce cyclic AMP synthesis, its stimulating effect on the release of CDD/ANP-(99–126) and the finding that the VIP-receptor antagonist inhibits responses to VIP but not to PACAP suggest that PACAP activates cardiac myocytes through a PACAP-specific receptor.