Radiofrequency ablation (RFA) in Brugada syndrome (BrS) has been performed by both endocardial and epicardial. The substrate in BrS is not completely understood. We investigate the functional endocardial substrate and its correlation with clinical, electrophysiological and ECG findings in order to guide an endocardial ablation. Two patients agreed to undergo an endocardial biopsy and the samples were examined with transmission electron microscopy (TEM) to investigate the correlation between functional and ultrastructural alterations. About 13 patients (38.7 ± 12.3 years old) with spontaneous type 1 ECG BrS pattern, inducible VF with programmed ventricular stimulation (PVS) and syncope without prodromes were enrolled. Before endocardial mapping, the patients underwent flecainide testing with the purpose of measuring the greatest ST-segment elevation for to be correlated with the size and location of substrate in the electro-anatomic map. Patients underwent endocardial bipolar and electro-anatomic mapping with the purpose of identify areas of abnormal electrograms (EGMs) as target for RFA and determine the location and size of the substrate. When the greatest ST-segment elevation was in the third intercostal space (ICS), the substrate was located upper in the longitudinal plane of the right ventricular outflow tract (RVOT) and a greatest ST-segment elevation in fourth ICS correspond with a location of substrate in lower region of longitudinal plane of RVOT. A QRS complex widening on its initial and final part, with prolonged transmural and regional depolarization time of RVOT corresponded to the substrate located in the anterior-lateral region of RVOT. A QRS complex widening rightwards and only prolonged transmural depolarization time corresponded with a substrate located in the anterior, anterior-septal or septal region of RVOT. RFA of endocardial substrate suppressed the inducibility and ECG BrS pattern during 34.7 ± 15.5 months. After RFA, flecainide testing confirmed elimination of the ECG BrS