Background Radiofrequency (RF) ablation has become a widely accepted treatment for atrial fibrillation (AF). This study aimed to identify the efficacy and safety of pulmonary vein (PV) ablation with ethanol and to explore an alternative energy source for catheter ablation of AF.Methods Twelve open-chest mongrel dogs were randomized into ethanol ablation group and control group. Both the injections and electrophysiological mapping procedures were performed epicardialy. In ethanol ablation group (n=6),injections were performed to circumferentially ablate the root of each PV (0.2 ml each site, 3 mm apart) with 95% ethanol using an 1 ml injector. In control group (n=-6), saline was injected other than ethanol. PV isolation was confirmed with a circular catheter immediately after the procedure and at follow up of 30 days. PV isolation was defined as the absence of PV potentials at each electrode of the circular catheter positioned at the PV side of the lesions, as well as complete conduction block into left atrium (LA) during PV pacing.Results PV electrical isolation with complete bidirectional conduction block was achieved with ethanol immediately and at 30 days in 95% of PVs, while saline injection caused only transient conduction changes between LA and PVs. In ethanol group, histologic analysis showed transmural lesions at 30 days. And there was no evidence of PV stenosis or thrombus formation. Mean LA diameter was not significantly different between baseline and 30 days.Conclusion Ethanol is a safe energy source to effectively isolate PV in canine model and may be promising in endocardial ablation procedure of AF patients in the future.