(1) Diadenosine polyphosphates have been described to be present in the myocardium and exert purinergic- and nonreceptor-mediated effects. Since the electrophysiological properties of atrial myocardium are effectively regulated by A(1) receptors, we investigated the effect of diadenosine pentaphosphate (Ap(5)A) in rabbit myocardium. (2) Parameters of supraventricular electrophysiology and atrial vulnerability were measured in Langendorff-perfused rabbit hearts. Muscarinic potassium current (I(K(ACh/Ado))) and ATP-sensitive potassium current (I(K(ATP))) were measured by using the whole-cell voltage clamp method. (3) Ap(5)A prolonged the cycle length of spontaneously beating Langendorff perfused hearts from 225+/-14 (control) to 1823+/-400 ms (Ap(5)A 50 micro M; n=6; P<0.05). This effect was paralleled by higher degree of atrio-ventricular block. Atrial effective refractory period (AERP) in control hearts was 84+/-14 ms (n=6). Ap(5)A>/=1 micro M reduced AERP (100 micro M, 58+/-11 ms; n=6). (4) Extrastimuli delivered to hearts perfused with Ap(5)A- or adenosine (>/= micro M)-induced atrial fibrillation, the incidence of which correlated to the concentration added to the perfusate. The selective A(1)-receptor antagonist CPX (20 micro M) inhibited the Ap(5)A- and adenosine-induced decrease of AERP. Atrial fibrillation was no longer observed in the presence of CPX. (5) The described Ap(5)A-induced effects in the multicellular preparation were enhanced by dipyridamole (10 micro M), which is a cellular adenosine uptake inhibitor. Dipyridamole-induced enhancement was inhibited by CPX. (6) Ap(5)A (</=1 mM) did neither induce I(K(Ado)) nor I(K(ATP)). No effect on activated I(K(Ado/ATP)) was observed in myocytes superfused with Ap(5)A. However, effluents from Langendorff hearts perfused with Ap(5)A 100 micro M activated I(K(Ado)) by using A(1) receptors. (7) Ap(5)A did not activate A(1) receptors in rabbit atrial myocytes. The Ap(5)A induced A(1)-receptor-mediated effects on supraventricular electrophysiology and vulnerability suggest that in the multicellular preparation Ap(5)A is hydrolyzed to yield adenosine, which acts via A(1) receptors. An influence on atrial electrophysiology or a facilitation of atrial fibrillation under conditions resulting in increased interstitial Ap(5)A concentrations might be of physiological/pathophysiological relevance.