The purpose of this study was to determine whether metabolites produced by glycolysis during ischemia significantly contribute to myocardial injury of hypertrophied hearts. The accumulation of glycolytic metabolites during ischemia was reduced by means of glycogen reduction or by treatment with the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG) before ischemia. Hearts from aortic-banded (Band) and sham-operated (Sham) rats (8 wk postop) were isolated, perfused with Krebs buffer, and had a left ventricular (LV) balloon to measure developed pressure. A 15-min perfusion with hypoxic buffer (glycogen reduction, GR) or a 10-min perfusion with 10 mM 2-DG (glycolytic inhibition) was followed by 25 min global, normothermic, no-flow ischemia and 30 min normoxic reperfusion. Heart weights were greater in Band than Sham [2.76 ± 0.06 vs. 1.5 ± 0.04 (means ± SE) g; P < 0.001]. GR and 2-DG each resulted in reduced ATP levels measured at the beginning of ischemia in both Band and Sham groups compared with untreated groups, but there were no differences among groups after 25 min of ischemia. Myocardial lactate levels at the end of ischemia were significantly reduced in both Band and Sham hearts with GR or 2-DG compared with untreated controls. Recovery of LV function after ischemia and reperfusion was significantly improved in Band after GR (206% increase) and after 2-DG treatment (126% increase) compared with their respective untreated controls. Diastolic dysfunction during reperfusion was ameliorated in Band by preischemic GR but not by 2-DG treatment. These findings support the hypothesis that glycolytic by-product accumulation during ischemia plays a significant role in injury of hypertrophied hearts after ischemia and reperfusion.