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The objective of the present study was to assess the degree to which astrocytic glutamine provides carbon for net synthesis of GABA in the rat neocortex in vivo. Isotopic labeling of GABA and glutamate from astrocytic glutamine was followed in halothane anesthetized and ventilated rats during an intravenous infusion of [2-¹³C]glucose. A net increase in GABA was achieved by administration of the GABA-transaminase inhibitor, gabaculine to suppress catabolism of GABA and recycling of ¹³C label. ¹³C Percentage enrichments of GABA, glutamate and glutamine were assessed in tissue extracts using ¹³C-edited ¹H nuclear magneic resonance at 8.4 T. GABA levels increased 2.6 μmol/g at 2 h and 6.1 μmol/g at 5 h after gabaculine, whereas glutamate and glutamine decreased in toto by 5.6 μmol/g at 2 h and 3.1 μmol/g at 5 h. Selective enrichment of glutamine, glutamate, and GABA C3’s over other carbon positions …