A simple relationship between parameters derived from a¹³C NMR isotopomer analysis and O₂ consumption is presented that allows measurement of the absolute rate of acetyl-CoA oxidation and anaplerotic flux in tissues oxidizing a mixture of four substrates. The method was first applied in a study of the effects of work state and β-adrenergic stimulation on net acetate oxidation and anaplerosis in the isolated working rat heart. The results demonstrate that the anticipated ratio of 2 between O₂ consumption and TCA cycle flux for hearts oxidizing only acetate holds at low workload when anaplerosis is low, but deviates toward a factor of 3 under high workload conditions when anaplerosis is increased. This analysis was also extended to hearts that oxidize a more physiological mixture of substrates including long-chain fatty acids, acetoacetate, lactate, pyruvate, and glucose. We show that the contribution each substrate makes to total TCA cycle flux can be determined by combined¹³C NMR and O₂ consumption measurements. The present study also demonstrates that stimulation of anaplerosis (by addition of propionate) can significantly alter the relative contribution each substrate makes to total TCA cycle flux. We conclude that if¹³C labeling patterns are selected appropriately, a comprehensive picture of flux through all major metabolic pathways feeding the cycle can be determined in a single experiment even when complex physiological mixtures of substrates are provided.