BACKGROUND The precise mechanism(s) governing the phenomenon of AV nodal Wenckebach periodicity is not fully elucidated. Currently 2 hypotheses, the decremental conduction and the Rosenbluethian step-delay, are most frequently used. We have provided new evidence that, in addition, dual pathway (DPW) electrophysiology is directly involved in the manifestation of AV nodal Wenckebach phenomenon. RESULTS AV nodal cellular action potentials (APs) were recorded from 6 rabbit AV node preparations during standard A1A2 and incremental pacing protocols. His electrogram alternans, a validated index of DPW electrophysiology, was used to monitor fast (FP) and slow (SP) pathway conduction. The data were collected in intact AV nodes, as well as after SP ablation. In all studied hearts the Wenckebach cycle started with FP propagation, followed by transition to SP until its ultimate block. During this process complex cellular APs were observed, with decremental foot formations reflecting the fading FP and second depolarizations produced by the SP. In addition, the AV node cells exhibited a progressive loss in maximal diastolic membrane potential (MDP) due to incomplete repolarization. The pause created with the blocked Wenckebach beat was associated with restoration of MDP and reinitiation of the conduction cycle via the FP wavefront. CONCLUSIONS DPW electrophysiology is dynamically involved in the development of AV nodal Wenckebach periodicity. In the intact AV node, the cycle starts with FP that is progressively weakened and then replaced by SP propagation, until block occurs. AV nodal SP modification did not eliminate Wenckebach periodicity but strongly affected its paradigm.