Administration of supplemental glucose and/or insulin is postulated to improve the outcome from myocardial ischemia by increasing the heart's relative utilization of glucose as an energy substrate. To examine the degree to which circulating glucose and insulin levels actually influence myocardial substrate preference in vivo, we infused conscious, chronically catheterized rats with D-[1-¹³C]glucose and compared steady-state ¹³C enrichment of plasma glucose with that of myocardial glycolytic ([3-¹³C]alanine) and oxidative ([4-¹³C]glutamate) intermediary metabolites. In fasting rats, [3-¹³C]alanine-to-[1-¹³C]glucose and [4-¹³C]glutamate-to-[3-¹³C]alanine ratios averaged 0.16 ± 0.12 and 0.14 ± 0.03, respectively, indicating that circulating glucose contributed 32% of myocardial glycolytic flux, whereas subsequent flux through pyruvate dehydrogenase contributed 14% of total tricarboxylic acid (TCA) cycle activity. Raising plasma glucose to 11 mmol/l, or insulin to 500 pmol/l, increased these contributions equivalently. At supraphysiological (>6,500 pmol/l) insulin levels, the plasma glucose contribution to glycolysis increased further, and addition of hyperglycemia made it the sole glycolytic substrate, yet [4-¹³C]glutamate-to-[3-¹³C]alanine ratios remained 0.60. Thus plasma levels of glucose and insulin independently regulate the proportional contribution of exogenous glucose to myocardial glycolytic and TCA cycle flux in vivo in a dose-dependent manner. However, even at supraphysiological levels, nonglucose substrates continue to supply 40% of myocardial TCA cycle flux.