Mitochondrial membrane potential oscillation was studied in isolated rat and guinea pig ventricular cardiomyocytes using fluorescence microscopy. Spontaneous oscillation of tetramethylrhodamine ethylester (TMRE) fluorescence occurred in the time scale of seconds; the oscillation was abolished by chelation of intracellular Ca²⁺. The TMRE fluorescence of each mitochondria unit changed independently, and a drop in TMRE fluorescence was preceded by a prolonged rise in fluo-4 fluorescence. Spontaneous oscillation of flavoprotein fluorescence in the second-to-minute time scale was observed in individual mitochondria units, which was abolished under chelation of intracellular Ca²⁺ and was followed by a decrease in TMRE fluorescence. The oscillation of TMRE fluorescence was markedly inhibited by inhibitors of mitochondrial electron transport or the permeability transition pore. These results indicate that fluctuations in intracellular Ca²⁺ and electron transport activity may cause fluctuations in mitochondrial membrane potential.