In the brain, glutaminase is considered to have a key role in the provision of glutamate, a major excitatory neurotransmitter. Brain slices obtained from wild-type (control) and glutaminase-deficient (GLS1 +/–) mice were incubated without glucose and with 5 or 1 mmol/L [3-¹³C]glutamine as substrate. At the end of the incubation, substrate removal and product formation were measured by both enzymatic and carbon 13 nuclear magnetic resonance (¹³C-NMR) techniques. Slices from GLS1 +/– mice consumed less [3-¹³C]glutamine and accumulated less [3-¹³C]glutamate. They also produced less ¹³CO₂ but accumulated amounts of ¹³C-aspartate and ¹³C-gamma-aminobutyric acid (GABA) that were similar to those found with brain slices from control mice. The newly formed glutamine observed in slices from control mice remained unchanged in slices from GLS1 +/– mice. As expected, flux through glutaminase in slices from GLS1 +/– mice was found diminished. Fluxes through all enzymes of the tricarboxylic acid cycle were also reduced in brain slices from GLS1 +/– mice except through malate dehydrogenase with 5 mmol/L [3-¹³C]glutamine. The latter diminutions are consistent with the decreases in the production of ¹³CO₂ also observed in the slices from these mice. It is concluded that the genetic approach used in this study confirms the key role of glutaminase for the provision of glutamate.