This study tests the hypothesis that a decrease of the free energy of ATP hydrolysis (Delta GATP) below a threshold value will inhibit Na+-K+-ATPase (Na+ pump) activity and result in an increase of intracellular Na+ concentration ([Na+]i) in the heart. Conditions were designed in which hearts were solely dependent on ATP derived from oxidative phosphorylation. The only substrate supplied was the fatty acid butyrate (Bu) at either low, 0.1 mM (LowBu), or high, 4 mM (HighBu), concentrations. Escalating work demand reduced the Delta GATP of the LowBu hearts. 31P, 23Na, and 87Rb NMR spectroscopy measured high-energy phosphate metabolites, [Na+]i, and Rb+ uptake. Rb+ uptake was used to estimate Na+ pump activity. To measure [Na+]i using a shift reagent for cations, extracellular Ca2+ was reduced to 0.85 mM, which eliminated work demand Delta GATP reductions. Increasing extracellular Na+ (Nae+) to 200 mM restored work demand Delta GATP reductions. In response to higher [Na+]e, [Na+]i increased equally in LowBu and HighBu hearts to approximately 8.6 mM, but Delta GATP decreased only in LowBu hearts. At lowest work demand the LowBu heart Delta GATP was -53 kJ/mol, Rb+ uptake was similar to that of HighBu hearts, and [Na+]i was constant. At highest work demand the LowBu heart Delta GATP decreased to -48 kJ/mol, the [Na+]i increased to 25 mM, and Rb+ uptake was 56% of that in HighBu hearts. At the highest work demand the HighBu heart Delta GATP was -54 kJ/mol and [Na+]i increased only approximately 10%. We conclude that a Delta GATP below -50 kJ/mol limits the Na+ pump and prevents maintenance of [Na+]i homeostasis.