Introduction: A prominent action potential (AP) notch in the epicardium (Epi) of the RVOT is known to predispose to the development of closely-coupled phase 2 reentrant extrasystoles, capable of precipitating ventricular tachycardia and fibrillation (VT/VF) in the setting of BrS. Ablation of this Epi substrate exerts an ameliorative effect. In some BrS patients, Endo ablation of the RVOT is effective as well. The prime objective of this study was to examine the electrophysiological basis for premature beats originating from the endocardium (Endo) of the right ventricular outflow tract (RVOT) in experimental models of Brugada syndrome (BrS). Methods: Canine coronary-perfused cardiac preparations incorporating the RVOT (n=15) were studied using standard microelectrode techniques. Terfenadine, a sodium and calcium channel blocker, was used to pharmacologically mimic the effects of the genetic defects associated with BrS. Results: Under baseline conditions, a prominent AP notch was recorded in Epi and in the deep intra-trabecular structures of RVOT Endo, but not in the smooth Endo surface of the RVOT. Terfenadine markedly accentuated the AP notch in the deep intra-trabecular structures of RVOT Endo leading to the development of closely-coupled phase 2 reentrant extrasystoles capable of triggering polymorphic VT/V. Still, Epi RVOT region was more likely to develop extrasystoles than Endo RVOT. VT/VF was recorded in 12/15 preparations. Conclusions: Our findings suggest that the deep intra-trabecular structures of RVOT Endo harbor the substrate for the development of phase 2 reentrant extrasystoles capable of triggering VT/VF. Our data may help to explain the effectiveness of Endo RVOT ablation in some BrS patients.