OBJECTIVE Atrioventricular node (AVN) modification is one of the alternatives for ventricular rate control in patients with drug refractory atrial fibrillation (AF). However, the underlying mechanisms, and in particular the role of the dual pathway electrophysiology is not clear. By using a novel index, His electrogram (HE) alternans, we have previously demonstrated in rabbits that both the slow (SP) and the fast pathways (FP) are involved in AVN conduction during AF. This electrophysiological-morphological study was designed to address the role of selective FP ablation on AVN conduction during AF. RESULTS In 12 rabbit AVN preparations dual pathway conduction was confirmed by HE alternans during A1A2 pacing protocol, as well as during AF. On average 48% of the conducted beats during AF utilized the FP. Selective FP ablation (n=12) guided by HE alternans resulted in only-SP conduction, with longer AVN conduction time at basic beats, but without change of AVN effective refractory period (ERP). Interestingly, despite elimination of all FP-conducted beats during AF, the selective FP ablation allowed previously concealed SP beats to be conducted, resulting in little net effect on the ventricular rate (average His-His interval 199+/-10 ms before versus 201+/-13 ms after FP ablation, p>0.05). Morphological evidence indicated that FP ablation created lesions within the transitional cells of the superior approaches at the junction between the central fibrous body and the AVN. However, extension of FP ablation lesion into the compact AVN domain resulted in non-selective AVN modification and slowing of ventricular rate during AF. CONCLUSIONS Despite its longer ERP, FP is responsible for a substantial number of ventricular beats during AF. However, selective FP ablation has a minor effect on ventricular rate. The most likely mechanism for this phenomenon is that FP ablation allows previously concealed SP beats to be conducted. On the other hand, ventricular rate slowdown could be achieved if FP ablations caused collateral damage in the compact node. This study highlights the usefulness of HE alternans as a novel tool to monitor dual pathway conduction during AF and to guide AVN modification.