Mathematical models of the TCA cycle derived previously for ¹⁴C tracer studies have been extended to ¹³C NMR to measure the ¹³C fractional enrichment of [2-¹³C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of ¹³C into the glutamate carbons. Hearts perfused with [2-¹³C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-¹³C]acetate. In each case, the amount of [2-¹³C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.