Background We previously demonstrated that papillary muscle (PM) can serve as an anchor of spiral waves in the in vitro preparations. Propranolol facilitates the anchoring. Whether or not the same is true in vivo is unclear. Methods and Results We used a high resolution (1792 channels, 1 mm inter-electrode distance) mapping system to study the epicardial activation during VF in 8 swine (25.1짹2.7 Kg) ventricles in vivo. A large electrode plaque containing 896 electrodes were placed on LV, over the root of papillary muscle (PM). The other electrodes were placed on RV and LV posterior wall. We also used the Endocardial Solutions Inc. multi-electrode array (>3000 virtual electrodes) to study endocardial activation in 2 of these swine. Data were acquired 7 to 15 s after electrical induction of VF at baseline and during propranolol infusion (0.05 mg/Kg bolus + 0.02 mg/Kg/min maintenance i.v.). Results: 1) Propranolol increased the incidence of stable epicardial reentry (SR: 6.9짹10.0 / 8sec vs. 3.2짹4.2 / 8 sec, p=0.002) and breakthrough (EB: 11.1짹13.9 / 8 sec vs. 5.0짹7.1 / 8 sec, p=1.4E-5) at anterior PM of LV. The life-spans of SR and EB were also lengthened (propranolol vs. baseline, 1.4짹1.8 sec vs. 0.5짹0.8 sec, p=0.001 and 2.2짹2.1 sec vs. 0.8짹1.1 sec, p<0.001, respectively). 2) Dominant frequencies (DF) of baseline VF were 9.4짹0.6 Hz, 10.1짹0.7 Hz and 10.4짹0.8 Hz, respectively (P<0.01) for RV, LV anterior wall, and LV posterior wall. However, there were no apicobasal or septo-free wall differences in DF. 3) Propranolol decreased the DF of VF and eliminated its regional heterogeneity (8.1짹1.1 Hz, 8.4짹1.1 Hz, 8.5짹1.3 Hz, respectively. P=NS). 4) Simultaneous endo- and epicardial mapping of VF in 2 swine showed that EB resulted from SR anchored to the PM. Conclusion Propranolol eliminates regional heterogeneity of activation rates and promotes stable spiral wave anchoring to the PM during VF in vivo.