The metabolic consequences of anoxia in the isolated, perfused guinea pig heart were examined by 13C NMR spectroscopy of 13C-labeled metabolites in situ. Upon addition of [3-13C]pyruvate to the perfusate during normoxic conditions, label is detected in several metabolites, including alanine (C3), glutamate (C2, C3, and C4), and aspartate (C2 and C3), reaching steady state levels 10-15 min after the labeled precursor reaches the heart. During anoxia, the label in glutamate and aspartate decreases and label appears in C2(3) of succinate. This real-time observation demonstrates that in the isolated intact heart, anaerobic metabolism of the amino acids aspartate and glutamate to succinate occurs. These pathways, which were first noted to occur in skeletal muscle of diving mammals, may provide a mechanism supplemental to glycolysis for the production of nucleoside triphosphates during periods of anoxia.