Cyclical changes in energy-related metabolites were observed in glucose-perfused but not pyruvate-perfused isolated working rat hearts. A chronological study of various phases of the cardiac cycle indicated maximum changes in metabolites occurred at half time to peak pressure (dF/dtmax). The high-energy phosphates ATP and phosphocreatine, as well as the glycolytic metabolites, glucose 6-phosphate and pyruvate, reached minimum values immediately prior to peak systole and maximum values during late diastole. The products of high-energy phosphate hydrolysis, ADP, inorganic phosphate, and creatine, as well as the regulator, adenosine 3',5'-cyclic monophosphate, showed the phase alternate. It was necessary to study cyclical changes in a maximally stressed glucose-perfused heart because the cyclical changes were small and appeared to be the result of rate-limiting steps in glycolysis and the slow transport of NADH into the mitochondria. For stressing the heart, thereby increasing ATP utilization and augmenting cyclical changes, the afterload chamber was set at 110 mmHg, and the perfusate contained high concentrations of calcium (3.5 mM, free) and isoproterenol (5 X 10(-9) M). When correction was made for binding and compartmentation of metabolites, data indicated that the free energy of ATP hydrolysis was preserved during the contraction process by a continuous binding and recycling of ADP.