Subcellular fractionation of tissue in nonaqueous media was employed to study metabolite compartmentation in isolated perfused rat hearts. The mitochondrial and cytosolic concentrations of citrate and 2-oxoglutarate, total concentrations of the glycolytic intermediates and rate of glycolysis were measured in connection with changes in the rate of cellular respiration upon modulation of the ATP consumption by changes of the mechanical work load of the heart. The concentrations of citrate and 2-oxoglutarate in the mitochondria were 16- and 14-fold, respectively, greater than those in the cytosol of beating hearts. The cytosolic citrate concentration was low compared with concentrations which have been employed in demonstrations of the citrate inhibition of glycolysis. In spite of the low activities reported for the tricarboxylate carrier in heart mitochondria, the cytosolic citrate concentration reacted to perturbations of the mitochondrial citrate concentration, and inhibition of glycolysis at the phosphofructokinase step could be observed concomitantly with an increase in the cytosolic citrate concentration. The delta pH across the inner mitochondrial membrane calculated from the 2-oxoglutarate concentration gradient and the mitochondrial membrane potential calculated from the adenylate distribution gave an electrochemical potential difference of protons compatible with chemiosmotic coupling in the intact myocardium.