Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by 13 C NMR and microdialysis combined with gas-chromatography/massspectrometry. Brain GLU C5 was 13 C enriched by intravenous [2,5-13 C]glucose infusion, followed by [ 12 C]glucose infusion to chase 13 C from the small glial GLU pool. This leaves [5-13 C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the 13 C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU ECF ) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter N enrichment of precursor NH 3 (0.87 ± 0.014), the rate of synthesis of GLN (V¢ GLN ), derived from neurotransmitter GLU ECF , was determined to be 6.4 ± 0.44 lmol/g/h. Comparison with V GLN measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 ± 0.44 lmol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU ECF , a process that is crucial for protecting the brain from GLU excitotoxicity.