BACKGROUND: Radiofrequency ablation of the mitral isthmus (MI) is unreliable. Vein of Marshall (VOM) ethanol infusion can improve MI ablation success and eliminate local MI autonomic innervation. The role of pulsed field ablation (PFA) in MI ablation and its autonomic effect is unclear. We evaluated the transmurality of endocardial PFA at the MI and its autonomic effects, using real-time epicardial VOM electrograms and VOM high-frequency stimulation. METHODS: Patients undergoing atrial fibrillation ablation (n=71) were prospectively enrolled. MI ablation (endocardial to VOM) was delivered using FaraPulse (N=31), PulseSelect (N=9), or Affera (N=31) while monitoring VOM epicardial electrograms to assess transmurality, defined as VOM signal elimination after 20 minutes. MI block was assessed using differential pacing from the left atrial appendage and VOM. High-frequency stimulation from the VOM was performed before and after PFA. VOM ethanol infusion was administered when MI ablation was incomplete. RESULTS: Endocardial PFA led to durable epicardial electrogram attenuation in 52.1% (FaraPulse in 38.7%, PulseSelect in 44.4%, and Affera in 67.7%; P<0.05). Myocardial capture with VOM pacing persisted in 43.7% (FaraPulse in 45.2%, PulseSelect in 100%, and Affera in 25.8%; P<0.05). Pseudoblock (epicardial-only MI conduction and MI delays) was present in 38%. MI block was achieved with PFA alone in 45% (32/71) of patients; VOM ethanol infusion achieved MI block in an additional 38% (27/71) of patients for a cumulative 83% (59/71); and coronary sinus ablation was required in the remaining 17% (12/71). After PFA, VOM high-frequency stimulation elicited intact parasympathetic responses evidenced as atrioventricular block. CONCLUSIONS: Across all 3 PFA platforms, endocardial ablation of the MI is not reliably transmural and does not ablate VOM parasympathetic innervation. VOM ethanol infusion can complete transmurality and denervation of the MI.