OBJECTIVE The goal of this study was to evaluate the effects of acute and chronic amiodarone on activation patterns during ventricular fibrillation (VF), ventricular effective refractory period (VERP) and defibrillation threshold (DFT). BACKGROUND Acute and chronic amiodarone may act through different mechanisms. METHODS The VERP, VF activation patterns and DFT were determined in 24 dogs. Twelve dogs received acute intravenous amiodarone (10 mg/kg, n = 6) or saline (n = 6), and 12 dogs received chronic oral amiodarone (20 mg/kg/day, n = 6) or placebo (n = 6). Epicardial VF activation patterns were recorded with 504 electrodes. Quantitative descriptors of VF were calculated. RESULTS The DFT was unchanged by acute or chronic amiodarone. Although chronic amiodarone significantly extended the VERP, acute amiodarone did not. In the mapped region, acute and chronic amiodarone decreased the number of VF wavefronts by 42% and 60%. Acute amiodarone decreased conduction block by 22%, while chronic amiodarone increased block by 41% but decreased wave fractionation by 50%. Both chronic and acute amiodarone increased the size of the core of re-entrant circuits and decreased the incidence of re-entry by 44% and 57%; however, chronic amiodarone increased wavelength, while acute amiodarone did not. CONCLUSIONS Neither acute nor chronic amiodarone change the DFT. While both acute and chronic amiodarone decrease the number of wavefronts, decrease the incidence of re-entry and increase the size of re-entrant cores in the mapped region during VF, they achieve these antiarrhythmic effects through different electrophysiologic mechanisms. Chronic amiodarone prolonged the VF cycle length and slowed conduction velocity, indicating it increased the wavelength and/or the excitable gap.