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Objective: : Clinical experience with endocardial cryoablation for the surgical treatment of atrial fibrillation has demonstrated safety and efficacy. Direct access to the left atrium via a thoracoscopic or pericardial approach with a balloon-tipped cryoablation catheter might facilitate endocardial cryoablation on the beating heart. We investigated the ability of a novel cryoballoon to produce endocardial pulmonary vein ostial cryolesions on the beating heart in a large-animal model., Methods: : Six sheep underwent small left thoracotomy. A 10.5F catheter with a 23-mm cryoballoon was inserted directly into the left atrium under fluoroscopic and intracardiac echo (ICE) guidance. Cryoablation of the pulmonary vein ostia was performed. Animals were killed at 14 days. Pulmonary venous electrical isolation was assessed immediately before the animals were killed., Results: : All animals survived balloon cryoablation with no periprocedural complications. Balloon occlusion was well tolerated hemodynamically, with minimal change in blood pressure (-4 ± 6 mm Hg systolic BP) and no change in heart rate. ICE demonstrated an absence of intracardiac air or ice embolization during ablation. Mean balloon temperature was -67 ± 8°C. All animals were neurologically intact after the procedure. Five of 6 (83%) veins exhibited circumferential exit block. Phrenic nerve function was intact in all animals. On gross inspection, all lesions were circumferential and continuous without evidence of endocardial thrombus. Pathology confirmed circumferential transmurality in all treated veins., Conclusions: : Direct left atrial access cryoballoon ablation was effective for isolating pulmonary veins. This technology may be an important component of a minimally invasive beating heart CryoMaze procedure for the treatment of atrial fibrillation.