This study examined the hypothesis that glycolysis is required for functional recovery of the myocardium during reperfusion by facilitating restoration of calcium homeostasis. [Ca2+]iwas measured in isolated perfused rabbit hearts by using the Ca2+indicator 1,2-bis(2-amino-5-fluorophenoxy)ethane-N,N,N',N'-tetraacetic acid (5F-BAPTA) and19F nuclear magnetic resonance spectroscopy. In nonischemic control hearts, inhibition of glycolysis with iodoacetate did not alter [Ca2+]i. In hearts subjected to 20 minutes of global zero-flow ischemia, [Ca2+]iincreased from 260±80 nM before ischemia to 556±44 nM after 15 minutes of ischemia (p<0.05). After reperfusion with 5 mM pyruvate as a carbon substrate, [Ca2+]iincreased further in hearts with intact glycolysis to 851±134 nM (p<0.05 versus ischemia) during the first 10 minutes of reperfusion, before returning to preischemic levels. In contrast, inhibition of glycolysis during the reperfusion period resulted in persistent severe calcium overload ([Ca2+]i, 1,380±260 nM after 15 minutes of reperfusion,p<0.02 versus intact glycolysis group). Furthermore, despite the presence of pyruvate and oxygen, inhibition of glycolysis during early reperfusion resulted in greater impairment of functional recovery (rate/pressure product, 3,722±738 mm Hg/min) than did reperfusion with pyruvate and intact glycolysis (rate/pressure product, 9,851±590 mm Hg/min,p<0.01). Inhibition of glycolysis during early reperfusion was also associated with a marked increase in left ventricular end-diastolic pressure during reperfusion (41±5 mm Hg) compared with hearts with intact glycolysis (16±2 mm Hg,p<0.01). The detrimental effects of glycolytic inhibition during early reperfusion were, however, prevented by initial reperfusion with a low calcium solution ([Ca]o, 0.63 mM for 30 minutes, then 2.50 mM for 30 minutes). In these hearts, the rate/pressure product after 60 minutes of reperfusion was 12,492±1,561 mm Hg/min (p<0.01 versus initial reflow with [Ca]oof 2.50 mM). These findings indicate that the functional impairment observed in postischemic myocardium is related to cellular Ca2+overload. Glycolysis appears to play an important role in restoration of Ca2+homeostasis and recovery of function of postischemic myocardium.