The role of lactate accumulation in lethal ischemic myocardial cell injury was assessed by partially depleting hearts of glycogen before ischemia by using glucagon. Isolated adult rat hearts were perfused with glucose-free Krebs-Henseleit buffer containing acetate as substrate. After stabilization, treated hearts were perfused briefly (3 min) with buffer containing 2 micrograms/ml glucagon to reduce tissue glycogen stores, followed by 10 min of perfusion with control buffer, and 60 or 90 min of global ischemia. Before the onset of ischemia, glucagon-treated hearts contained 40% less glycogen than untreated hearts, but myocardial function and tissue levels of high-energy phosphates, lactate, and glucose 6-phosphate were similar. Lactate production during ischemia in the glucagon-treated hearts was 50% less than in untreated hearts. However, there was no decrease in the amount of creatine kinase release during reperfusion after either 60 or 90 min of ischemia. Thus although partial glycogen depletion reduced lactate accumulation during ischemia, this did not decrease the amount of lethal myocardial cell injury.