Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ-aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore we measured 13C incorporation into brain metabolites by dynamic 13C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α-chloralose anaesthesia during infusion of [1,6-13C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of 13C into the three aliphatic carbons of GABA non-invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11±0.01 μmol/g/min. GABA-glutamine cycle was 0.053±0.003 μmol/g/min and accounted for 22±1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47±0.02 μmol/g/min, of which 35±1% and 7±1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12±1% of the TCA cycle flux was dedicated to oxidation of GABA. 16±2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain. This article is protected by copyright. All rights reserved.