Thyroid hormone regulates metabolism through transcriptional and post-transcriptional mechanisms. The integration of these mechanisms in heart is poorly understood. Therefore, we investigated control of substrate flux into the citric acid cycle (CAC) by thyroid hormone using retrogradely perfused isolated hearts (n=20) from control (C) and age-matched thyroidectomized rats (T). We determined substrate flux and fractional contributions (Fc) to the CAC by ¹³C NMR spectroscopy and isotopomer analyses in hearts perfused with 1,3-[¹³C]acetoacetic acid (0.17 mM), L-lactic-3-[¹³C] acid (LAC 1.2 mM), U-[¹³C]-long chain mixed free fatty acids (FFA, 0.35 mM) and unlabeled glucose. Some T hearts were supplied triiodothyronine (T3, 10 nM) (TT) for 60 min. Prolonged hypothyroid state reduced myocardial oxygen consumption, although T3 produced no significant change. Hypothyroidism reduced overall CAC_flux but selectively altered only FFA_flux among the individual substrates, though LAC_flux trended upwards. T3 rapidly decreased lactate Fc and flux. ¹³C labeling of glutamine through glutamate was increased in T with further enhancement in TT. The glutamate/glutamine ratio was significantly lower in T and TT. Immunoblots detected a decrease in hypothyroid hearts for muscle carnitine palmitoyl transferase I (CPTI) and a marked increase in pyruvate dehydrogenase kinase (PDK) 2 with no changes in liver-CPTI, PDK4 or hexokinase 2. TT, though not T, displayed elevated glutamine synthetase (GS) expression. These studies showed that T3 regulates cardiac metabolism through integration of several mechanisms including changes in oxidative enzyme content and rapid modulation of individual substrates fluxes. T3 also moderates forward glutamine flux, possibly by increasing the overall activity of GS.