Atrial fibrillation (AF) is a complex disease with multiple mechanisms, involving the interaction between the autonomic nervous system (ANS), electrophysiological properties of the atria and pulmonary veins (PVs), and vulnerability for AF. OBJECTIVE We assessed the effects of acute vagal (vagus_stim) and sympathetic stimulation (symp_stim) on atrial conduction, atrial and PV refractoriness and inducibility of AF in an in vivo rabbit model with preserved autonomic innervation. METHODS An open-epicardial approach was used in 17 anesthetized and artificially ventilated New Zealand white rabbits. The ECG was recorded with bipolar subcutaneous electrodes placed in the four limbs. Electrograms were obtained with four monopolar electrodes placed epicardially along the atria, and a circular electrode adapted to the proximal PVs. The cervical vagus nerve and thoracic sympathetic trunk were stimulated with bipolar electrodes. Epicardial activation was recorded in sinus rhythm, and effective refractory periods (ERPs) and conduction times from the high-lateral right atrium (RA) to the high-lateral left atrium (LA) and PVs were quantified at baseline and during vagus_stim, symp_stim, or combined vagal and sympathetic stimulation (dual_stim). Burst pacing (50 Hz, 10 s), alone or combined with vagus_stim, symp_stim or dual_stim, was performed in the right (RAA) and left atrial appendage (LAA) and PVs to test for AF inducibility. RESULTS At baseline, ERPs were higher in the LAA and there was a delay in the conduction time from RA to PV, compared to the mean activation time from RA to LA. During vagus_stim or dual_stim, ERP decreased significantly at all sites, and baseline interatrial activation times changed from 20 +/- 4 ms to 30 +/- 10 ms and 31 +/- 11 ms, respectively (p < 0.05). Symp_stim resulted in a significant decrease in ERPs only in the LAA, and a reduction of the interatrial interval to 16 +/- 11 ms (p < 0.05 vs baseline). AF inducibility ranged from 35% to 53% with baseline 50 Hz pacing, 65% to 76% during vagus_stim or symp_stim, and 75% to 100% with dual_stim (p < 0.05). AF duration increased significantly during ANS stimulation. In two-thirds of the animals with longer inducible AF, the arrhythmia ceased immediately after cessation of vagus_stim. CONCLUSIONS In the fully innervated rabbit heart in vivo, acute ANS stimulation shortens atrial and PV refractoriness, and significantly changes atrial conduction times, promoting AF induction and prolonging the arrhythmia. This underscores the importance of acute variations in ANS tone and its interactions in the pathophysiology of AF.