Background - It is believed that defibrillation is due to shock-induced changes of transmembrane potential (ΔVm) in the bulk of ventricular myocardium (so-called virtual electrodes), but experimental proof of this hypothesis is absent. Here, intramural shock-induced ΔVm were measured for the first time in isolated preparations of left ventricle (LV) by an optical mapping technique. Methods and Results - LV preparations were excised from porcine hearts (n=9) and perfused through a coronary artery. Rectangular shocks (duration 10 ms, field strength E ≊ 2 to 50 V/cm) were applied across the wall during the action potential plateau by 2 large electrodes. Shock-induced ΔVm were measured on the transmural wall surface with a 16X16 photodiode array (resolution 1.2 mm/diode). Whereas weak shocks (E≊2 V/cm) induced negligible ΔVm in the wall middle, stronger shocks produced intramural ΔVm of 2 types. (1) Shocks with E>4 V/cm produced both positive and negative intramural ΔVm that changed their sign on changing shock polarity, possibly reflecting large-scale nonuniformities in the tissue structure; the ΔVm patterns were asymmetrical, with ΔV-m>ΔV+m. (2) Shocks with E>34 V/cm produced predominantly negative ΔVm across the whole transmural surface, independent of the shock polarity. These relatively uniform polarizations could be a result of microscopic discontinuities in tissue structure. Conclusions - Strong defibrillation shocks induce ΔVm in the intramural layers of LV. During action potential plateau, intramural ΔVm are typically asymmetrical (ΔV-m>ΔV+m) and become globally negative during very strong shocks.