The authors have previously shown that ischemia causes inhibition of the respiratory chain and phosphorylation system, and stimulation of the proton leak of mitochondria isolated from rat heart. It is shown here that the activity of the mitochondrial respiratory chain (after 30 min ischemia, but not after 45 min) and the phosphorylation system are completely restored to the normal level by the addition of exogenous cytochrome c when succinate is used as substrate. Moreover, cytochrome c causes apparent activation of the respiratory chain, the phosphorylation system and the proton leak in normal mitochondria. This can be explained by a fraction of the mitochondrial population lacking cytochrome c and this fraction may increase with ischemia. Experiments on skinned cardiac fibers showed that cytochrome c has no effect on mitochondrial respiration after 15 min ischemia, but the stimulation of respiration by cytochrome c progressively increases when ischemia was prolonged up to 30 min and 45 min, suggesting that the loss of cytochrome c may occur in vivo during the early reversible phase of ischemia. Mitochondria isolated from hearts after 45 min ischemia have a defect in the respiratory chain unrelated to the loss of cytochrome c. These mitochondria have an increased level of Ca2+: 10.05 nmol/mg protein compared to 4.64 nmol/mg in control mitochondria and 4.32 nmol/mg in 30 min ischemic mitochondria. The increase in the proton leak in ischemic mitochondria is mostly reversible by albumin and thus, may be related to an increased level of free fatty acids in ischemic mitochondria.