The effects of 11.7 mM glucose, insulin, and potassium (GIK) on metabolism during ischemia were investigated in the perfused guinea pig heart using magnetic resonance spectroscopy. Intracellular metabolites, primarily glycogen and glutamate, were labeled with 13C by addition of [1-13C]glucose to the perfusate during a normoxic, preischemic period. 13C and 31P NMR spectroscopy was used to observe the metabolism of 13C-labeled metabolites simultaneously with high-energy phosphorus metabolites and pH. The extent of acidosis and the rate and amount of labeled lactate accumulation during ischemia were the same in control (3 mM glucose + insulin) and GIK-treated hearts. In contrast, the rate of labeled glycogen mobilization during ischemia in GIK-treated hearts was one third the rate observed in control hearts. These observations suggest that GIK decreased the rate of glycogenolysis during ischemia without affecting the rate of glycolysis. We propose that glucose contributed as a glycolytic substrate to a greater extent during ischemia in GIK-treated hearts than in hearts perfused with 3 mM glucose and insulin. The glycogen-sparing effect of GIK demonstrated in these studies could delay the onset of ischemic damage in a clinical setting by prolonging the availability of glycolytic substrate necessary for production of high-energy phosphate.