Both phases of the calcium paradox were associated with major alterations in myocardial energy metabolism. During calcium-free perfusion contractility of the heart ceased, resulting in a dramatic decrease in anaerobic and aerobic metabolism but no change in tissue high energy phosphate levels. Tissue content of most citric acid cycle intermediates were elevated, while there was a net decrease in the content of transaminase-linked amino acids. Reperfusion of the calcium-depleted heart with calcium-containing buffer failed to restore either the contractile or the metabolic state of the heart. Within seconds following calcium repletion, tissue high energy phosphate content plummeted. This occurred even though glucose utilization increased significantly and aerobic metabolism remained at levels observed in the calcium-depleted heart. Analogous to changes seen in acidosis and ischemia, alpha-ketoglutarate and citrate levels decreased abruptly. After a short delay, the levels of several key amino acids also dropped. The results support the hypothesis that the impairment of mitochondrial function contributes to the depletion of high energy phosphate stores during the calcium paradox.