A new 13C NMR technique for measuring substrate utilization by the citric acid cycle based on an analysis of succinate 13C isotopomers is presented. The relative contribution of up to three different labeling patterns in acetyl-CoA entering the citric acid cycle may be determined under non-steady-state conditions. We present experimental data from perfused rat hearts subjected to a brief period of ischemia, where both succinate and glutamate resonances were observed in the 13C spectrum. The contributions of labeled exogenous acetate and lactate and unlabeled sources to the acetyl-CoA pool were compared using this succinate analysis and a previously published glutamate analysis [Malloy et al. (1990) Biochemistry 29, 6756-6761], and the two methods give identical results. This indicates that the succinate and glutamate isotopomers originated from a common alpha-ketoglutarate pool, verifying that glutamate is in isotopomeric equilibrium with alpha-ketoglutarate under these conditions.