Little is known about the three-dimensional (3-D) intramural activation sequences during long-duration ventricular fibrillation (VF), including the role of the subendocardium and its Purkinje fibers (PFs) in long-duration VF maintenance. Our aim was to explore the mechanism of long-duration VF maintenance with 3-D electrical mapping. We recorded 10 min of electrically induced VF in the left ventricular anterior free wall of six 10-kg, open-chest dogs using a 3-D transmural unipolar electrode matrix (9 x 9 x 6, 2-mm spacing) that allowed us to map intramural activation sequences. At 2.5 ± 1.8 min of VF, although the body surface ECG continued to exhibit a disorganized VF pattern, intramurally a more organized, synchronous activation pattern was first observed [locally synchronized VF (LSVF)]. This pattern occurred one or more times in all dogs and was present 33.4 ∼ 31.4% of the time during 5-10 min of VF. As opposed to the preceding changing complex activation sequences of VF, during LSVF, wavefronts were large and highly repeatable near the endocardium, first exciting the endocardium almost simultaneously and then rapidly spreading toward the epicardium with different levels of conduction block en route. During LSVF, PF activations always preceded working myocardium activations near the endocardium. In conclusion, long-duration VF in dogs frequently becomes highly organized in the subendocardium, with activation fronts arising in this region and passing intramurally toward the epicardium, even though the surface ECG continues to exhibit a disorganized pattern. PFs appear to play an important role during this stage of VF. Copyright © 2010 the American Physiological Society.