We have recently demonstrated that during pacing-induced sustained ventricular fibrillation (VF), perfusion of the heart with either ruthenium red (RR) or Ru 360, blockers of the mitochondrial Ca2+ uniporter, resulted in the reversible conversion of VF to ventricular tachycardia (VT). Here, we aimed at elucidating the electrophysiological mechanisms for the RR-induced conversion of VF to VT. The experiments were performed using Langendorff-perfused isolated rat hearts in which left ventricular pressure and left ventricular intracellular action potential were recorded. Perfusion with either RR or Ru 360 resulted in decreases in the action potential duration (APD), refractory period, and slope of APD restitution curves. These changes were antagonized by cotreatment with S(-)-Bay K8644. In addition, perfusion with verapamil produced the decreases in APD at 90% repolarization, refractory period and slope of APD restitution curves similar to the RR or Ru 360 perfusion. Such electrophysiological changes may be responsible for the reversible conversion of sustained VF to VT caused by perfusion with RR or Ru 360.