Entry of 13C-enriched acetyl-CoA into the citric acid cycle results in scrambling of 13C into the various carbon positions of all intermediate pools. The eventual result is that the 13C resonances of all detectable intermediates or molecules exchanging with those intermediates appear as multiplets due to nearest neighbor spin-spin couplings. We have previously shown that an isotopomer analysis of the glutamate 13C multiplets provides a history of 13C flow through the cycle pools and that relative substrate utilization and relative anaplerotic flux can be quantitated (C.R. Malloy, A.D. Sherry, and F.M.H. Jeffrey, Am. J. Physiol. 259, H987-H995 (1990)). A major limitation of the method for in vivo applications is spectral resolution of multiline resonances required for a complete isotopomer analysis. We now show that [13C]homonuclear decoupling of the glutamate C3 resonance collapses nine-line C4 and C2 resonances into three-line multiplets. We demonstrate that these three-line 13C multiplets are well resolved in isolated, perfused rat hearts and present steady-state equations that allow an isotopomer analysis from data obtained in intact tissue. This advancement offers for the first time the possibility of extending 13C isotopomer methods to complex metabolic conditions in vivo.