Background: The preferential or fastest internodal pathways(INP), which impulses travel from the sinus to AV node(AVN), have been contested. This study were performed to evaluate the characteristics of INPs, the change of preferential INPs after sympathetic stimulation, and the relationship between earliest activation site(EAS) and INPs. Methods: Using 3D endocardial mapping, the conduction via INP and EAS were evaluated in 60 atrial fibrillation(AF) patients without sinus dysfunction(48 men, 57.9짹9.8 years) during conscious anesthesia. Anterior, middle and inferior INPs were defined as the tracts travel via the Bachmann bundle, behind superior vena cava, and along the crista terminalis(CT) to the AVN, respectively. Results: At baseline, 50(83%) patients had unicentric EAS. Among these patients, the location of EAS was superior, middle and lower parts of CT in 25(50%), 20(40%) and 5(10%) patients, respectively. The preferential INP was anterior, middle and inferior INP in 29(58%) and 17(34%) and 3(6%) patients, respectively. The conduction time of each INP was 43.3짹16.1ms, 48.7짹12.9ms, 71.6짹15.8ms, respectively. During isoproterenol infusion of 7-10 關g/min, 44(90%) patients had unicentric EAS at superior, middle and lower CT in 38(86%), 2(5%) and 4(9%) patients, respectively. The anterior, middle and inferior INP was the fastest INP in 35(85%), 2(5%) and 4(10%) patients, respectively. Compared with baseline, more patients had superior CT as the EAS(p<0.001) and anterior INP as the preferential INP(p=0.002) after isoproterenol infusion. Interestingly, all patients having the superior CT as the EAS at baseline(n=25) and during isoproterenol infusion(n=35) had the anterior INP as the fastest INP. Conclusions: The preferential INPs were observed in most patients. During sympathetic stimulation, the EAS was located at the superior CT, and the anterior INP was the fastest one. The preferential INP was closely associated with the location of EAS, suggesting that the location of INP was controlled by EAS.