The collapse of mitochondrial inner-membrane potential (ΔΨm) is a key determinant of cell injury and arrhythmogenesis associated with ischemia-reperfusion or oxidative stress. We have previously demonstrated that mitochondrial uncoupling resulting from the activation of energy-dissipating mitochondrial channels can activate sarcolemmal KATP channels, which can profoundly alter cellular electrical excitability. We proposed that these regions could serve as “metabolic sinks” of current that could be a substrate for reentry. Here, we directly test whether local ΔΨm loss influences electrical propagation in monolayers of neonatal rat ventricular myocytes (NRVMs) using a method of local perfusion of a portion of the monolayer with the mitochondrial oxidative phosphorylation uncoupler, FCCP (a protonophore; 1μM). Propagation of the electrical wave through the monolayer was recorded by optical mapping with a 464-element photodiode array using the voltage-sensitive fluorescent dye, di-4-ANEPPS. Using a custom-built perfusion device, a 5mm circular zone in the center of the coverslip (full diameter 2cm) was exposed to FCCP with the remainder perfused with normal Tyrode's buffer, and the monolayer was stimulated from one edge at 1Hz. Upon encountering the metabolic sink, the wave of electrical depolarization was slowed and the amplitude of the action potentials in the FCCP-perfused area decreased significantly. The results indicate that heterogeneous ΔΨm collapse can significantly alter the electrical substrate in a manner that could promote reentry.