Abstract Background Cardioneuroablation (CNA) treats reflex syncope by ablating the main epicardial ganglionated plexuses (GPs) conveying vagal outputs to the sino-atrial (SA) and atrio-ventricular (AV) nodes. The two main GPs, superior paraseptal (SPSGP) and inferior paraseptal (IPSGP), regulate vagal tone to SA and AV nodes, respectively. Both GPs can be targeted with radiofrequency energy (RF) from the right (RA) and left atrium (LA). It is unclear if bi-atrial ablation yields more effective GP ablation or if the ablation sequence affects GP response. Purpose We aimed to assess the added efficacy of ablating the SPSGP and IPSGP in the LA after initial RA ablation, and vice versa, in a separate group of patients. Methods In a consecutive patient population undergoing CNA we evaluated the acute efficacy of RF delivery on the SPSGP and the IPSGP. The population was divided into two consecutive groups: one receiving RF initially in the RA and then in the LA (group 1), and the other in the reverse order (group 2). The acute effect of RF applications was measured as percentage increase in heart rate (HR) for the SPSGP and as reduction of PR interval for the IPSGP. Results Sixty-one patients (50±15 years; 61% males) underwent CNA. Of these, 17 underwent ablation of the SPSGP and IPSGP starting from the RA and completing the procedure in the LA (group 1), while in 44 patients, the procedure was performed in the opposite order (group 2). In group 1, RF delivery on the SPSGP from the RA prompted an increase in HR (54.1±11.1 vs 60.6±14.7 bpm; p=0.03; median HR increment 3.8%, IQR 0-38.3) that was further enhanced after LA ablation (from 57.5±11.9 to 64.5±13.5 bpm; p<0.001; median HR increment: 8.0%, IQR: 2.0-13.5). In group 1, RA ablation of the IPSGP reduced PR interval in a single patient (from 224 to 184 ms), without further shortening after LA ablation. Final CNA success was 82% in group 1. In group 2, RF delivery on the SPSGP from the LA induced a marked increase in HR (58.4±13.1 vs 76.8±16.1 bpm; p<0.001; median HR increase 30.0%, IQR 14.0-43.0). In the same patients, additional ablation from the RA prompted further but lower incremental effect (from 70.0±17.3 to 76.9±17.9 bpm; p<0.001; median HR increment 8.3%, IQR: 0-16.4). In group 2, 4 (9%) patients had PR shortening after LA ablation of the IPSGP (241±93 vs 172±14 ms; p value). Of these, a single patient had further PR reduction after IPSGP RA ablation (from 189 to 161 bpm). Final CNA success was 93% in group 2. Symptoms-free survival was 88% at 16±5 months in group 1 and 89% at 19±7 months in group 2 (p= ns). Conclusions Bi-atrial ablation of the SPSGP and IPSGP provides incremental acute efficacy compared to ablation from either the RA or the LA alone. LA ablation of the main GPs seems an essential component of CNA. The physiological significance of these findings requires further investigationGanglionated Plexuses Segmentation