Absolute metabolic fluxes in isolated perfused hearts have been determined by a nonlinear least squares analysis of glutamate labeling kinetics from [1- 13C]glucose, [4-13C]β-hydroxybutyrate, or [2-13C]acetate using 13C NMR spectroscopy. With glucose as substrate, the malate-aspartate shuttle flux was too slow to account for the reducing equivalents generated by glycolysis and to predict the observed oxygen consumption rate. For acetate and β-hydroxybutyrate, the malate-aspartate shuttle had to be reversed for the network to agree with the observed oxygen consumption and glutamate labeling. Thus, an additional redox shuttle was required to reoxidize the NADH produced by cytoplasmic malate dehydrogenase. Using this model there was good agreement between the experimentally determined oxygen consumption and glutamate labeling and the calculated values of these parameters from the model for all substrates. The contribution of exogenous substrate to the overall tricarboxylic acid (TCA) cycle flux, 89.6 ± 6.5% (mean ± S.D.) as measured in the tissue extracts compared well with 91.4 ± 4.2% calculated by the model. The ratio of TCA cycle flux to oxygen consumption for acetate, was 2.2 ± 0.1, indicating that NADH production is principally accounted for by TCA cycle flux. For glucose or β-hydroxybutyrate, this ratio was 2.9 ± 0.2, consistent with the existence of other NADH producing reactions (e.g. glycolysis, β-hydroxybutyrate oxidation).