Because the electrophysiological effects of pituitary adenylate cyclase-activating polypeptide (PACAP) on the heart are little known, we studied the regulation of the atrial ATP-sensitive K⁺ (K_ATP) current by PACAP on primary cultured neonatal rat atrial myocytes. PACAP-38 stimulates cAMP production with EC₅₀ = 0.28 nmol/l (r = 0.92, P < 0.02). PACAP-38 and PACAP-27 (10 nmol/l) have similar maximal effects, whereas 100 nmol/l vasoactive intestinal polypeptide (VIP) is 2.7 times less effective (P < 0.05). RT-PCR shows the presence of cloned PACAP receptors PAC₁ (2 isoforms), VPAC₁, and VPAC₂. PACAP-38 dose dependently activates the whole cell atrial K_ATP current with EC₅₀ = 1-3 nmol/l (n = 44). Maximal effects occur at 10 nmol/l (91 ± 15 pA/pF, n = 18). Diazoxide further increases the PACAP-activated current by 78% (P < 0.05; n = 6). H₈₉ (500 nmol/l), a protein kinase A (PKA) inhibitor, reduces the PACAP-activated K_ATP current to 17.8 ± 9.6% (n = 5) of the maximal diazoxide-induced current and totally inhibits the cAMP-induced K_ATP current. A protein kinase C (PKC) inhibitor peptide (50 µmol/l) in the pipette reduces the PACAP-38-induced K_ATP current to 33 ± 17 pA/pF (P < 0.05, n = 6) without significantly affecting the currents induced by cAMP or VIP. The results suggest that: 1) PAC₁, VPAC₁, and VPAC₂ are present in atrial myocytes; and 2) PACAP-38 activates the atrial K_ATP channels through both PKA and PKC pathways.