An alternative 13C NMR method which allows direct determination of substrate oxidation in tissue for up to three competing 13C-enriched substrates is presented. Oxidation of competing substrates can be measured by 13C NMR spectroscopy under non-steady-state conditions if the relative areas of the glutamate C3 and C4 resonances can be determined. The accuracy of this measurement is limited during brief exposure to 13C-enriched substrates because of the low enrichment in the C3 carbon. The glutamate C4 resonance from a tissue sample which has oxidized a combination of [1,2-13C]acetate (or a uniformly enriched fatty acid mixture) and [3-13C]lactate appears as a nine-line resonance consisting of four multiplet components: a singlet (C4S), two doublets with differing one-bond coupling constants (C4D34 and C4D45), and a quartet (C4Q). It is shown that the sum of the C4S + C4D34 resonance areas versus the C4D45 + C4Q resonance areas directly reports the relative utilization of [3-13C]lactate versus [1,2-13C]acetate, respectively, regardless of citric acid cycle intermediate pool sizes or carbon flux through anaplerotic reactions. We also show that homonuclear 13C decoupling of the glutamate C2 resonance collapses the C3 resonance multiplet into an apparent triplet (actually, a singlet plus a doublet); the relative area of the singlet component reflects the amount of unlabeled acetyl-CoA entering the cycle. The method has been used to determine the contribution of lactate/acetate/glucose to acetyl-CoA in normoxic and reperfused rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)