The effect of hypothermia of 25° C for 24 hours on myocardial metabolism and efficiency was determined on dogs fasted for approximately 15 hours and anesthetized with sodium pentobarbital. Coronary blood flow, cardiac output, myocardial oxygen and substrate utilization, and mechanical efficiency of the heart were determined at normal and reduced body temperatures. Prolonged reduction of myocardial temperature, with concomitant reduction in coronary blood flow, led to diminished oxygen and substrate utilization. Myocardial glycolysis began following 12 hours of cooling when pyruvate utilization stopped in negative balance. After 24 hours the heart stopped utilizing carbohydrates, with negative arteriovenous differences for these substrates (in the presence of normal arterial carbohydrate levels), but continued to utilize nonesterified fatty acid. The coefficient of oxygen utilization for the heart increased following 24 hours of cooling, suggesting a relative state of myocardial hypoxia. The appearance of hypoxia and glycolysis during the late hours of cooling suggest that the heart's limit of tolerance to cooling was near.