This study investigates the generation of hydrogen peroxide (H₂O₂) by pigeon heart mitochondria and its dependence on various factors such as substrates, antimycin A, pH, and oxygen pressure. The maximal rate of H₂O₂ production by pigeon heart mitochondria is about 20 nmol/min per mg of protein, with succinate-glutamate and malate-glutamate as effective substrates. Antimycin A significantly enhances H₂O₂ production, and the effect is dependent on the energy state of the mitochondrial membranes. The generation of H₂O₂ is linearly related to the amount of incorporated ubiquinone, and the optimal pH for H₂O₂ production varies depending on the metabolic state and the presence of antimycin A. Under hyperbaric oxygen conditions, H₂O₂ production increases significantly, with rates increasing up to 10-15-fold in rat liver mitochondria. The study also suggests that H₂O₂ generation may be due to an energy-dependent component of the respiratory chain at the cytochrome b level. The results indicate that mitochondrial H₂O₂ generation is a physiological event under aerobic conditions and is influenced by various metabolic factors. The study highlights the importance of understanding the mechanisms of H₂O₂ generation in mitochondria, as it has implications for cellular oxygen toxicity and the physiological role of catalase in regulating intracellular H₂O₂ levels.This study investigates the generation of hydrogen peroxide (H₂O₂) by pigeon heart mitochondria and its dependence on various factors such as substrates, antimycin A, pH, and oxygen pressure. The maximal rate of H₂O₂ production by pigeon heart mitochondria is about 20 nmol/min per mg of protein, with succinate-glutamate and malate-glutamate as effective substrates. Antimycin A significantly enhances H₂O₂ production, and the effect is dependent on the energy state of the mitochondrial membranes. The generation of H₂O₂ is linearly related to the amount of incorporated ubiquinone, and the optimal pH for H₂O₂ production varies depending on the metabolic state and the presence of antimycin A. Under hyperbaric oxygen conditions, H₂O₂ production increases significantly, with rates increasing up to 10-15-fold in rat liver mitochondria. The study also suggests that H₂O₂ generation may be due to an energy-dependent component of the respiratory chain at the cytochrome b level. The results indicate that mitochondrial H₂O₂ generation is a physiological event under aerobic conditions and is influenced by various metabolic factors. The study highlights the importance of understanding the mechanisms of H₂O₂ generation in mitochondria, as it has implications for cellular oxygen toxicity and the physiological role of catalase in regulating intracellular H₂O₂ levels.