Endocardial mapping has suggested that Purkinje fibers may play a role in the maintenance of long duration ventricular fibrillation (LDVF). To determine the influence of Purkinje fibers on LDVF, we chemically ablated the Purkinje system with Lugol solution and recorded endocardial and transmural activation during LDVF. Dog hearts were isolated and perfused, and the left ventricular endocardium was exposed and treated with Lugol solution (n=6) or normal Tyrode solution as a control (n=6). The anterior papillary muscle endocardium was mapped with a 504 electrode (21x24) plaque with electrodes spaced 1 mm apart. Transmural activation was recorded with a 6-electrode plunge needle on each side of the plaque. VF was induced, and perfusion was halted. LDVF spontaneously terminated sooner in Lugol ablated hearts than in control hearts (4.9 ± 1.5 min vs. 9.2 ± 3.2 min, p=0.01). After termination of VF, both the control and Lugol hearts were typically excitable, but only short episodes of VF could be reinduced. Endocardial activation rates were similar during the first 2 min of LDVF for Lugol ablated and control hearts, but were significantly slower in Lugol hearts by 3 min. In control hearts, the endocardium activated more rapidly than the epicardium after 4 min of LDVF with wavefronts propagating most often from the endocardium to epicardium. No difference in transmural activation rate or wavefront direction was observed in Lugol hearts. Ablation of the subendocardium hastens VF spontaneous termination and alters VF activation sequences, suggesting that Purkinje fibers are important in the maintenance of LDVF.