Preconditioning decreases ischemic injury, preserves tissue ATP content, and enhances the salutary effects of reperfusion. To evaluate whether preserved ATP is related to reduced utilization or increased production, 28 paced isolated rabbit hearts, perfused at constant flow, were subjected to 3 min of transient no-flow ischemia followed 12 min later by 1 h of low-flow ischemia and 45 min of reperfusion. Results were compared with those from 34 control hearts subjected to ischemia and reperfusion without preconditioning. Preconditioning delayed the onset of ischemic contracture and decreased its amplitude. At the end of ischemia, tissue ATP content was higher in hearts subjected to preconditioning (9.8 +/- 3.3 vs. 4.5 +/- 1.1 mumol/g dry wt; P < 0.01), accounted for by increased anaerobic ATP production using exogenous glucose. Preconditioning decreased ischemic damage (creatine kinase release 373 +/- 199 vs. 587 +/- 291 U/g dry wt; P < 0.05) and resulted in better functional recovery with reperfusion (74 +/- 11% of baseline developed pressure vs. 60 +/- 23%; P < 0.05). Thus preconditioning appears to protect ischemic myocardium by enhancing anaerobic glycolytic production of ATP using exogenous glucose.