We investigated by C-13 NMR the turnover of the H3 deuterons of (2-C-13) glutamate and (2-C-13) glutamine in the brain of partially deuterated rats. Adult animals (150-200 g) fed ad libitum received 50%(H2O)-H-2 or tap water 9 days before infusing (1-C-13) glucose or (2-C-13) acetate for 5, 10, 15, 30, 60, or 90 min. The brains were then funnel-frozen and acid extracts were prepared and analyzed by high-resolution C-13 NMR. The deuteration of one or the two H3 hydrogens of (2-C-13) glutamate or glutamine resulted in single (-0.07 ppm) or double (-0.14 ppm) isotopic shifts upfield of the corresponding C2 perprotonated resonance, demonstrating two sequential deuteration steps. The faster monodeuteration generated 3R or 3S (2-C-13, 3-H-2) glutamate or glutamine through the alternate activities of cerebral aconitase or isocitrate dehydrogenase, respectively. The slower process produced bideuterated (2-C-13, 3,3'-H-2(2)) glutamate or glutamine through the consecutive activity of both enzymes. The kinetics of deuteration was fitted to a Michaelis-Menten model including the apparent K-m(') and V-max(') values for the observed deuterations. Our results revealed different kinetic constants for the alternate and consecutive deuterations, suggesting that these processes were caused by the different cytosolic or mitochondrial isoforms of aconitase and isocitrate dehydrogenase, respectively. The deuterations of (2-C-13) glutamate or glutamine followed also different kinetics from (1-C-13) glucose or (2-C-13) acetate, revealing distinct deuteration environments in the neuronal or glial compartments.