The glutathione in mitochondria is thought to play a crucial role in protecting mitochondria against ischemia-reperfusion-induced injury. However, little is known about the mitochondrial redox system. This study was conducted to clarify changes of mitochondrial glutathione redox during liver ischemia and reperfusion and its role on energy producing function. Rats were divided into three groups each of which were treated respectively with saline (Control), buthionine sulfoximine (BSO), which induces a rapid decrease in tissue glutathione concentrations, and gamma-glutamylcysteine ethyl ester (GCE), which conversely induces a rapid increase in tissue glutathione concentrations before induction of ischemia. Liver ischemia was induced for 120 min, and blood reflow was subsequently restored for 60 min. Total and mitochondrial glutathione concentrations, mitochondrial respiratory function, and tissue adenine nucleotide were determined after both the ischemic and the reperfusion periods. In all groups, concentrations of the reduced form of glutathione (GSH) gradually decreased during ischemia and reperfusion. On the other hand, significant increases in mitochondrial GSH were apparent after reperfusion despite significant decreases during ischemia in the control and GCE groups. Total and mitochondrial GSH in the BSO and GCE groups were significantly lower and higher, respectively, compared with the control throughout the experiment. Recovery of the mitochondrial energy producing function and cellular adenine nucleotide after reperfusion were dependent on GSH concentrations. We conclude that mitochondrial GSH concentrations dramatically change in a different manner from cytosolic concentrations after reperfusion, and that recovery of the mitochondrial energy-producing function might be closely associated with mitochondrial GSH concentrations.