Background-The presence of multiple arrhythmogenic sources may be associated with the perpetuation of atrial fibrillation (AF). In this study, we investigated the hypothesis that multiple foci might be involved in the development of AF persistency. Methods and Results-Two hundred fourteen consecutive patients with AF undergoing catheter ablation were enrolled in this study. The location of the arrhythmogenic foci was determined using simultaneous recordings from multipolar catheters before and after pulmonary vein isolation during an isoproterenol administration. We detected 500 arrhythmogenic foci (263 foci as AF initiators, and 237 foci as non-AF initiators). High-dose isoproterenol infusions (ranging from 2 to 20 g/min) revealed potential arrhythmogenic foci, especially non-pulmonary vein foci (55%). Persistent AF was more highly associated with an incidence of multiple (Ͼ2) foci than paroxysmal AF (88% versus 65%, Pϭ0.002), and a multivariate analysis demonstrated that multiple foci (Ͼ2) were an independent contributing factor for persistent AF (odds ratio; 95% confidence interval, 4.69; 1.82 to 12.09, PϽ0.001). In paroxysmal AF, the number of foci was higher in patients with long-term AF (Ͼ24 hours) than in those with short-lasting AF (2.64Ϯ0.14 versus 1.77Ϯ0.16, Pϭ0.001). In the persistent AF group, the patients with short-lasting AF (Ͻ12 months) had a greater number of foci than did those with long-term AF (Ͼ12 months) (3.62Ϯ0.15 versus 1.92Ϯ0.16, Pϭ0.04). Conclusions-Multiple foci were likely to be involved in the development of persistent AF. However, if AF persisted for Ͼ12 months, they may not have had a significant effect on the AF perpetuation. (Circ Arrhythm Electrophysiol. 2010; 3:39-45.)