The 13C-label incorporation into glutamate, glutamine, aspartate and gamma-aminobutyric acid (GABA) from [2-13C] glucose was measured by 13C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [2-13C] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of 13C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The 13C entered the TCA cycle via pyruvate carboxylase from [2-13C] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate > GABA > glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the 13C-skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.