beta-Hydroxy-butyrate (HBA) is an effective substrate for mitochondrial respiration (MVO2) in the heart. Myocardial HBA oxidation is associated with high mitochondrial NADH and an inhibition of glycolytic flux. The purpose of this study was to investigate if the infusion of HBA in vivo could modify the coupling mechanisms between myocardial MVO2 and work in the presence and absence of epinephrine. The extraction of several oxidized metabolites, O2, and HBA was measured during the infusion of HBA as well as the high-energy phosphate metabolites using 31P-nuclear magnetic resonance spectroscopy. HBA infusion did not affect the MVO2 or function of the heart with or without epinephrine infusion. However, HBA increased the phosphorylation potential by decreasing inorganic phosphate and the calculated free ADP concentration under both conditions. This is consistent with HBA increasing the mitochondrial NADH, which results in an increase in the phosphorylation potential without modifying function. These data demonstrate that substrates, specifically HBA, can modulate the cardiac phosphorylation potential in vivo. The most likely mechanism for this effect is through the mitochondrial NADH concentration.