During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca²⁺ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, β, γ-methylene adenosine 5′-triphosphate, prevented both the release of Ca²⁺ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A₂, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca²⁺ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A₂ suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca²⁺, which activate phospholipase A₂, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H⁺ of the inner membrane. The results also indicate another event; activation of phospholipase A₂ by release Ca²⁺ which results in H⁺ leakiness of the inner membrane.