Objective: Glycolysis-from-glucose may be more beneficial than glycogenolysis in protecting hearts against ischemia. We tested the hypothesis that ischemic preconditioning is mediated by increased exogenous glucose use during low-flow ischemia, an effect triggered by adenosine A₁ receptor activation. Methods: Langendorff rat hearts were subjected to 25 min low-flow ischemia (0.6 ml/min) and 30 min reperfusion. Prior to underperfusion, hearts (n=6 per group) were subjected to two cycles of either preconditioning ischemia (PC), infusion of the adenosine A₁ agonist 2-chloro-N⁶-cyclopentyladenosine (CCPA; 0.25 μmol/l), or PC in the presence of the adenosine antagonist 8-(p-sulfophenyl)theophylline (SPT; 50 μmol/l). Glycolysis-from-glucose during underperfusion was measured using d-[2-³H]glucose. Results: At the end of reperfusion, recovery of rate-force product was enhanced in the PC and CCPA groups (62 and 67% of preischemic value) compared to the ischemic control hearts (IC, 32%; P<0.05), whereas protection was abolished in the SPT hearts (20%; P<0.05 vs. PC). PC improved total glycolysis-from-glucose during underperfusion by 31% (P<0.05 vs. IC); SPT abolished this increase. CCPA reduced total lactate release and glucose uptake during ischemia by 47% and 61%, respectively (P<0.05 vs. IC). Abolishment of the preconditioning-associated increase in glucose uptake during underperfusion, by switching to a low glucose buffer, resulted in a loss of functional protection. Conclusions: This study strongly suggests that increased exogenous glucose utilization during low-flow ischemia mediates ischemic preconditioning without increasing total anaerobic glycolytic flux. Although adenosine A₁ receptor activation reduces ischemic injury, it does not facilitate the increased glucose uptake observed with ischemic preconditioning, suggesting a different mechanism of protection.