BACKGROUND: The purpose of this study was to test whether radiofrequency catheter ablation (RFCA) of 3 epicardial fat pads that resulted in efferent vagal denervation of the atria and sinus and atrioventricular nodes also denervated the ventricles. METHODS AND RESULTS: Vagal innervation of the ventricles was determined by measuring prolongation of ventricular effective refractory period induced by bilateral vagal stimulation (20 Hz, 10 V, 4 ms). Changes in heart rate variability (HRV) and baroreflex sensitivity (BRS) were also examined. We found that RFCA of the 3 epicardial fat pads vagally denervated the sinus and AV nodes and atria without affecting vagal innervation of the ventricles, indicating that efferent vagal fibers to the ventricles do not travel through the 3 epicardial fat pads. Parameters of time-domain variables decreased significantly; the total-power, high-frequency, and low-frequency components of frequency-domain variables decreased significantly; and the ratio of the low- and high-frequency components increased significantly after chronic vagal denervation. Vagally modulated sinus arrhythmia and BRS were also eliminated after chronic vagal denervation. These data also indicate that HRV and BRS represent vagal activity at the level of the sinus node and may not accurately reflect efferent vagal activity at the ventricular level. CONCLUSIONS: Selective vagal denervation of the sinus and AV nodes and atria decreased HRV and eliminated BRS while preserving ventricular innervation.