Technical Abstract: Brain glutamic acid (GLU) and gamma aminobutyric acid (GABA) derive virtually exclusively from synthesis de novo. Although glucose is the main source of their carbon skeleton, the source of nitrogen remains controversial. Because leucine (LEU) is readily transported into the brain and the brain contains both cytosolic and mitochondrial branched-chain aminotransferase (BCAT), we hypothesized that LEU is the critical nitrogen precursor for brain GLU synthesis. Rats (177 +/- 8g) were given hourly meals containing U13C- and/or 15N-LEU. Blood and tissue samples were harvested at 0.5, 1, 2, 3, 6, 9 hr of feeding (2-6 rats per time). 15N-and U13C-enrichments in plasma and brain LEU, GLU, GABA, and glutamine (GLN) were measured by GCMS. Plasma and brain LEU equilibrated very rapidly, the ratio reaching steady state in less than 30 min. Sequestration of 15N within the brain was compatible with a LEU uptake of 0.8-1 micro mol/g/h. The isotopic dilution of U13C (brain: plasma ratio 0.66 +/- 0.06) and 15N LEU (brain plasma ratio 0.26 +/- 0.02) differed markedly, suggesting that 13% of the brain LEU flux derived from proteolysis and 61% from cyclic transamination. On the basis of the kinetics of 15N-brain LEU and GLU labeling, brain LEU turnover was 45% per min, brain GLU synthesis via BCAT was 1.9 micro mol/g/h, and more than 50% of the GLU-N derived from leucine. The 15N-enrichments of GLU (5.1 +/- 0.3%), aspartic acid (5.4 +/- 0.3%) and GABA (5.5 +/- 0.4%) were higher (p<0.05, p<0.001, p<0.01, respectively, paired t-test) than that of the amino nitrogen of GLN (4.9 +/- 0.2%). The higher 15N-enrichment in GABA suggests that LEU directly supplies nitrogen to the neuronal metabolic pools as well as to the glial pool.