Euthanasia is the humane termination of an animal's life and an important consideration for scientists, veterinarians, regulators, and others contemplating investigations involving animals. Techniques for euthanasia must induce the most rapid, painless, and distress-free death possible. This study investigated the effectiveness of direct current induction of ventricular fibrillation for the euthanasia of sheep after a primary study in which artifacts or chemical contamination from injectable euthanasia agents were undesirable. Female crossbred adult sheep (Ovis aries; n = 12) under deep isoflurane general anesthesia were instrumented with electrophysiology catheters to induce ventricular fibrillation for euthanasia. Data regarding invasive arterial blood pressure, expired airway gases, limb lead electrocardiograms, and pulse oximetry were collected and assessed just prior to, immediately after, and at 5, 10, 15, and 20min after energy delivery. In all animals, a single 10-s application of 9V of direct current to the right ventricular endocardium via the electrophysiology catheter induced persistent ventricular fibrillation. Arterial blood pressure (mean ± 1 SD) immediately after fibrillation induction was 22.9±4.5mmHg, with negligible difference between systolic and diastolic pressures. The lack of differential pressure continued through the end of the monitoring period. Arterial blood pressure reached an initial nadir at 1??0.5min after fibrillation induction, peaked (40.8±11.1mmHg) due to a vasoconstrictive reflex at 3min after induction, and returned to a static uniform pressure (20.4±17.8mmHg) with mildly increased variability due to reflexive diaphragmatic contractions at 10min after induction. The use of 9V direct current for the induction of ventricular fibrillation via an electrophysiology catheter is a reliable method of euthanasia in sheep.