Interatrial connection (IAC) is reported to be an anatomical structure associated with complex biatrial tachycardias. However, electrophysiological conduction between left atrium (LA) and right atrium (RA) has not been fully investigated. This study was to distinguish multiple IACs by using different pacing sites. Twenty patients with paroxysmal atrial fibrillation who underwent ablation were enrolled. High-density mapping of left and right atrial before ablation were created during continuous pacing from sinus node (SN) or low lateral RA (LL-RA), and LA appendage (LAA) or posterior wall (LA-PW), respectively. Focal activation sites were defined as IAC and breakthrough segments of each atrium were recorded. The average number of total IAC were 4.9±1.3 and 4.1±1.2 for RA-LA and LA-RA, respectively (Figure A1,2). There was no significant difference in number of IACs between each directional connections (cranial pacing, p=0.35; caudal pacing, p=0.62) (Figure B). Connections at the level of interatrial septum (IAS) and Bachmann’s bundle were mostly found as RA-LA connections, however more than one third of the patients had connections in right pulmonary veins, its carina, LA roof and coronary sinus musculature. LA-RA connections were mainly existed in IAS, however superior and inferior vena cava were also frequently related. Only either single site from SN or LL-RA pacing elucidated significantly smaller number of IACs for RA-LA compared to total IACs (SN vs total, 3.2±0.8 vs 4.9±1.3, p<0.001; LL-RA vs total, 3.2±1.5 vs 4.9±1.3, p<0.001) (Figure C1). Similarly, only either LAA or LA-PW pacing revealed significantly smaller number of IACs for LA-RA compared to total IACs (LAA vs total, 3.0±0.8 vs 4.1±1.2, p<0.001; LA-PW vs total, 3.0±0.8 vs 4.1±1.2, p<0.001) (Figure C2). Although difference of pacing sites did not influence the total number of interarial conductions (RA-LA, p=0.09; LA-RA, p=0.85), pacing from cranial and caudal sites preferentially disclosed IAC at upper and lower septal segments, respectively (upper connections of RA-LA, SN vs LL-RA, 73.4% vs 46.9%, p=0.0037; upper connections of LA-RA, LAA vs LA-PW pacing, 66.1% vs 41.0%, p=0.013) (Figure D1,2). Pacing from multiple locations may be required in order to reveal entire interatrial connections.