Metabolism of [2-13C]-, [3-13C]-, and [1,2,3-13C]propionate in perfused rat livers and [2-13C]-acetate in perfused rat hearts has been examined in tissue extracts by 13C NMR. Label from [2-13C]-propionate was preferentially incorporated into the C2 carbon of lactate, alanine, and aspartate in liver tissue while label from [3-13C]propionate appeared preferentially in the C3 carbon of those same molecules. These data suggest that 13C may not be completely randomized in the symmetric citric acid cycle intermediates succinate and fumarate as is normally assumed but that some fraction of those intermediates may be transferred between enzymes in this span of the cycle with conservation of spatial orientation, consistent with recent results obtained in yeast [Sumegi et al. (1990) Biochemistry 29, 9106-9110]. This was confirmed by performing similar experiments with [1,2,3-13C]propionate. Time-dependent asymmetry was also observed between the intensities of the glutamate C2 and C3 resonances and between the aspartate C2 and C3 resonances in 13C NMR spectra of intact hearts and heart extracts during early perfusion with [2-13C]-acetate. A model is presented which predicts that isotopic asymmetry is observed only during the first 2-3 turns of the cycle pools when isotope enters the cycle via acetyl-CoA even if all symmetric cycle intermediates retain a unique molecular orientation on each pass through the citric acid cycle.