Although the tricarboxylic acid (TCA) cycle is the prime means of carbon metabolism for energy generation in normal myocardium, the noninvasive quantification of TCA cycle flux in intact cardiac tissues is difficult. A novel approach for estimating citric acid cycle flux using13C nuclear magnetic resonance (NMR) is presented and evaluated experimentally by comparison with measured myocardial oxygen consumption over a wide range of cardiac contractile function in intact, beating rat hearts. Continuous series of13C NMR spectra, obtained after the introduction of [2-13C] acetate as substrate, quantified the time course of13C appearance in the carbon positions of myocardial glutamate, which are sequentially enriched via citric acid cycle metabolism. A TCA cycle flux parameter was calculated using the premise that TCA cycle flux is inversely proportional to the time difference between13C appearance in the C-4 and C-2 positions of glutamate (glutamate δt50[minutes]), which are enriched in subsequent “turns” of the TCA cycle. This TCA cycle flux parameter, termed KT, correlated strongly with myocardial oxygen consumption over a range of developed pressures in hearts perfused with 5 mM acetate (r=0.98,p<0.001), as well as in separate studies in hearts perfused with 5 mM glucose and 0.5–0.8 mM acetate (r=0.94,p<0.001). Results of numerical modeling of13C glutamate kinetics suggest that this TCA cycle flux parameter, KT, is relatively insensitive to changes in metabolite pool sizes that could occur during metabolism of other substrates or during conditions of altered oxygen availability. Additional studies in separate hearts indicated that the time course of13C appearance in citrate, which is predominantly mitochondrial in the rat heart, is similar to that in glutamate, further supporting the premise that the described13C NMR parameters reflect mitochondrial citric acid cycle activity in intact cardiac tissues.