Aspartate aminotransferase (AAT) catalyzes amino group transfer from glutamate (Glu) or aspartate (Asp) to a keto acid acceptoroxaloacetate (OA) or -ketoglutarate (KG), respectively. Data presented here show that AAT catalyzes two partial reactions resulting in isotope exchange between ³H-labeled Glu or ³H-labeled Asp and the cognate keto acid in the absence of the keto acid acceptor required for the net reaction. Tritiated keto acid product was detected by release of ³H₂O from C-3 during base-induced enolization. Tritium released directly from C-2 (or C-3) by the enzyme was also evaluated and is a small fraction of that released because of exchange to the keto acid pool. Exchange is dependent on AAT concentration, time-dependent, proportional to the amino-to-keto acid ratio, and blocked by aminooxyacetate (AOA), an AAT inhibitor. Enzymatic conversion of [³H]KG to Glu by glutamic dehydrogenase (GDH) or of [³H]OA to malate by malic dehydrogenase (MDH) "protects" the label from release by base, showing that base-induced isotope release is from keto acid rather than a result of release during the exchange process. AAT isotope exchange is discussed in the context of the glutamate/glutamine shuttle hypothesis for astrocyte/neuron carbon cycling.