Background: Knowledge of transmural Vm changes is important for understanding the mechanism of long-duration ventricular fibrillation (LDVF). Objective: The purpose of this study was to measure transmural Vm changes during LDVF. Methods: Vm was recorded optically at up to 8 transmural points separated by 1.5 mm in the left ventricle of Langendorff-perfused canine hearts (n = 6) using a bundle of optical fibers (optrode) during 10 minutes of LDVF followed by 3 minutes of VF with reperfusion. Measurements were grouped into 4 layers: epicardium, subepicardium, midwall, and subendocardium. Results: Activation rates (ARs) and action potential durations (APDs) decreased, whereas diastolic intervals (DIs) increased during LDVF in all transmural layers (P < .05). After approximately 3 minutes of LDVF, ARs were faster and DIs shorter in the midwall and subendocardium than in the epicardium and subepicardium (P < .05). Activations persisted at the subendocardium but disappeared from other layers after approximately 8 minutes of VF in the majority of hearts. There were no transmural differences in APD during LDVF or during pacing before and after LDVF (P > .05). Restitution plots showed no functional relationship between APD and DI in any layer at any stage of LDVF. Partial reperfusion during VF for 3 minutes restored transmural synchronicity of activation and eliminated gradients in activation parameters. Conclusion: Vm dynamics evolve differently at different transmural layers. The subendocardium maintains persistent and the fastest activation during 10 minutes of LDVF, suggesting it contains the source of VF wavefronts. There are no transmural APD gradients and no restitution relationship between APD and DI at any transmural layer, indicating these are not the primary factors in the mechanism of LDVF. © 2009 Heart Rhythm Society.