Background and Objective: Ectopic beats originating from the pulmonary vein (PV) trigger atrial fibrillation (AF). The purpose of this study was to clarify the electrophysiological determinant of AF initiation from the PVs. Methods: Pacing studies were performed with a single extra stimulus mimicking an ectopic beat in the left superior pulmonary veins (LSPVs) in 62 patients undergoing AF ablation. Inducibility of AF, effective refractory period (ERP) and conduction properties within the PVs were analyzed. Results: A single extra stimulus in LSPV induced AF in 20 patients (32% of all patients) at the mean coupling interval (CI) of 172 ms. A CI-dependent anisotropic conduction at the AF onset was visualized in a 3D-mapping. Onset of AF was site-specific with reproducibility in each individual. Mean ERP in LSPV in the AF inducible group was shorter than that in the AF non-inducible group (182 ± 55 ms vs 254 ± 51 ms, P<0.0001). LSPV ERP dispersion was greater in the AF inducible group than in the AF non-inducible group (45 ± 28 ms vs 27 ± 19 ms, P<0.01). Circumferential intra-PV conduction time (IPVCT) exhibited decremental properties in response to shortening of CI, and the prolongation of IPVCT in the AF inducible site was greater than that in the AF non-inducible site (P<0.05) in each individual. Conclusions: Location and coupling interval of an ectopic excitation ultimately determine the initiation of AF from the PVs. ERP dispersion and circumferential conduction delay may lead to anisotropic conduction and reentry within the PVs that initiate AF.