Hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) of rat brain mitochondria is associated with membrane regions thought to correspond to contact sites (regions of close interaction of the inner and outer mitochondrial membranes). Two intramitochondrial compartments of ATP also appear to be located at contact sites, and are dependent on oxidative phosphorylation for their generation. Neither of these compartments was associated with the intermembranal space containing adenylate kinase, nor was there detectable intramitochondrial compartmentation of ATP generated by the adenylate kinase reaction. Formation of these compartments was not dependent on the presence of bound hexokinase since equivalent amounts of compartmented ATP were found in mitochondria from which a major portion of the hexokinase had been removed by treatment with Glc-6-P. During active oxidative phosphorylation, mitochondrially bound hexokinase is totally dependent upon intramitochondrially compartmented ATP as a substrate. Both the levels of ATP in the intramitochondrial compartments and the rate of glucose phosphorylation by mitochondrially bound hexokinase were shown to be correlated with the rate of oxidative phosphorylation. This dependence of hexokinase on intramitochondrial ATP levels that reflect the status of mitochondrial oxidative metabolism provides a mechanism by which hexokinase can serve as a mediator, coordinating the rate at which glucose is introduced into the glycolytic pathway with terminal oxidative stages of metabolism and avoiding the accumulation of lactate which has been associated with toxic effects on the brain.