Reactive oxygen species (ROS)-induced ROS release (RIRR) is a novel phenomenon observed in cardiac myocytes during the mitochondrial permeability transition (MPT). This study demonstrates that ROS accumulation in individual mitochondria, triggered by photoactivation of tetramethylrhodamine derivatives, leads to abrupt mitochondrial depolarization and MPT induction. The process is prevented by the ROS scavenger Trolox and is accompanied by increased mitochondrial ROS production, termed RIRR. This phenomenon occurs synchronously and reversibly among adjacent mitochondria, suggesting a cooperative mechanism. The MPT induction triggers RIRR, which is linked to the collapse of the mitochondrial membrane potential (ΔΨ) and the release of ROS. The study shows that ROS, particularly peroxides, are key initiators of MPT and RIRR, with the MPT pore allowing the passage of molecules up to 1.5 kD. The findings suggest that ROS-induced MPT and RIRR are fundamental processes in mitochondrial and cell biology, with potential implications for cardiac function and disease. The study also highlights the role of glutathione in ROS scavenging and the importance of redox state in regulating MPT. The results indicate that MPT induction is essential for the ROS burst, and that the MPT pore operates in a single open state, capable of flickering between open and closed states. The study provides insights into the mechanisms of MPT and its regulation by ROS, with potential implications for understanding cardiac pathophysiology and the development of therapeutic strategies.Reactive oxygen species (ROS)-induced ROS release (RIRR) is a novel phenomenon observed in cardiac myocytes during the mitochondrial permeability transition (MPT). This study demonstrates that ROS accumulation in individual mitochondria, triggered by photoactivation of tetramethylrhodamine derivatives, leads to abrupt mitochondrial depolarization and MPT induction. The process is prevented by the ROS scavenger Trolox and is accompanied by increased mitochondrial ROS production, termed RIRR. This phenomenon occurs synchronously and reversibly among adjacent mitochondria, suggesting a cooperative mechanism. The MPT induction triggers RIRR, which is linked to the collapse of the mitochondrial membrane potential (ΔΨ) and the release of ROS. The study shows that ROS, particularly peroxides, are key initiators of MPT and RIRR, with the MPT pore allowing the passage of molecules up to 1.5 kD. The findings suggest that ROS-induced MPT and RIRR are fundamental processes in mitochondrial and cell biology, with potential implications for cardiac function and disease. The study also highlights the role of glutathione in ROS scavenging and the importance of redox state in regulating MPT. The results indicate that MPT induction is essential for the ROS burst, and that the MPT pore operates in a single open state, capable of flickering between open and closed states. The study provides insights into the mechanisms of MPT and its regulation by ROS, with potential implications for understanding cardiac pathophysiology and the development of therapeutic strategies.