Background Ischemic preconditioning (IPC) attenuates acidosis during prolonged ischemia and improves contractile and metabolic parameters during subsequent reperfusion. Glycogen depletion induced by IPC is proposed as a potential mechanism. Methods and Results We studied the influence of manipulations of preischemic glycogen levels (Pre-G, μmol glucose/g wet wt) on contractile and metabolic (via 31 P–nuclear magnetic resonance) parameters during 30 minutes of ischemia and recovery in four groups of isovolumic rat hearts: First, control (Con, n=18, mean Pre-G, 21.5±0.8); second, after two 5-minute IPC periods (IPC, n=12, Pre-G, 11.3±0.7); third, a control group in which Pre-G was depleted by glucose-free, acetate perfusion (Con-LowG, n=9, Pre-G, 7.9±1.2); and fourth, an IPC group in which Pre-G was raised by glucose and lactate perfusion such that Pre-G was similar to Con (IPC-HiG, n=11, Pre-G, 20±1.4). Manipulation of Pre-G significantly altered the pH fall during 30 minutes of ischemia (Con, 5.76±.03, Con-LowG, 6.26±.07; IPC-HiG, 5.91±.02, IPC, 6.05±.09). IPC-HiG hearts had significantly worse metabolic recovery (PCr, 70±7 versus 91±3% initial; IPC-HiG versus IPC, P <.05) and contractile recovery (end-diastolic pressure, 52±5 versus 29±5 mm Hg, P <.05) than IPC hearts but better recovery than Con (%PCr, 56±6% and end-diastolic pressure, 72±6 mm Hg). An ischemic rise in intracellular magnesium occurred and was atttenuated in preconditioned hearts. Conclusions Pre-G levels before ischemia influence but are not the sole determinants of the extent of acidosis during prolonged ischemia and of metabolic and contractile recovery during reperfusion in control and preconditioned hearts.