To investigate mitochondrial regulation and its response to a defect in oxidative metabolism, we used 31P-magnetic resonance spectroscopy to study phosphocreatine (PCr) recovery in rat leg muscle after sciatic nerve stimulation at 1-4 Hz. We studied normal animals and animals with defective skeletal muscle mitochondrial function after experimental cardiac infarction. To analyze these data, we used three current theoretical approaches to the control of mitochondrial ATP synthesis, based on its hyperbolic relationship to cytosolic ADP concentration and on its linear relationships to PCr concentration and the free energy of ATP hydrolysis. The mitochondrial ADP concentration for one-half maximum rate of ATP synthesis appeared at least twice as high as the 30 microM expected from in vitro studies. According to all three approaches, the apparent maximum rate of ATP synthesis was independent of stimulation frequency and end-exercise pH and PCr and ADP concentrations and was reduced by approximately 50% after experimental cardiac infarction. Analysis of PCr recovery kinetics is a robust and practical way to study mitochondrial regulation and to quantify effective mitochondrial defects in vivo.