The nature of the metabolic events leading to irreversible damage of the ischemic myocardium are not known. We tested the hypothesis that catabolism of adenosine triphosphate precursors limits the regeneration of this high-energy compound following ischemic insult. Dogs on cardiopulmonary bypass (CPB) had the aorta cross-clamped for 20 minutes at normothermia and 30 minutes later CPB was discontinued. Mean pre-CPB values (μmoles/gm) in left ventricular biopsies for ATP, creatine phosphate (CP), ADP, and AMP were 5.30, 6.18, 1.32 and 0.43, respectively. Adenosine (Ad), inosine, and hypoxanthine/xanthine were not detectable. At the end of cross-clamping, ATP had fallen to 2.80 and CP to 0.57; however, the predicted rise in the levels of ADP, AMP, and Ad was not found. The levels of inosine (1.23) and hypoxanthine/xanthine (0.24) increased, an indication that ADP, AMP, and Ad had been further catabolized. Following release of the cross-clamp, ATP levels did not increase; even 60 minutes after termination of CPB this level was only 3.07. CP levels rose to 10.2 within 5 minutes after cross-clamp release, indicating high-energy bonds could be formed. EHNA (10 mg/kg), an inhibitor of adenosine deaminase, was used to test the effect of blocking Ad catabolism. At the end of cross-clamping, tissue Ad, previously unmeasurable, was 1.30 μmoles/gm. Nevertheless, AtP levels did not rebound after release of the cross-clamp; the reason was found to be cellular loss, and the coronary sinus blood contained high levels of Ad. Ad (20 mg/kg) infusion alone increased myocardial blood flow fivefold compared to flow in control dogs, but did not alter subsequent ATP levels. Combined EHNA/Ad treatment similarly increased flow but, more importantly, resulted in recovery of ATP levels to 88% of pre-CPB values. We conclude that ATP regeneration after ischemia is limited by the availability of ADP, AMP, and Ad. Inhibition of Ad catabolism and infusion of Ad will enhance ATP return from ischemia. Current preservation methods, chiefly hypothermia and cardioplegia, are designed to decrease ATP utilization. Our approach, by providing precursors for ATP recovery, may further improve myocardial preservation.