Glutamate is the major excitatory neurotransmitter, and is inactivated by cellular uptake catalyzed mostly by the glutamate transporter subtypes GLT‐1 (EAAT2) and GLAST (EAAT1). Astrocytes express both GLT‐1 and GLAST, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we injected GLT‐1 knockout (KO) mice and wild‐type littermates with 1‐13Cglucose and 1,2‐13Cacetate 15 min before euthanization. Metabolite levels were analyzed in extracts from neocortex and cerebellum and 13C labeling in neocortex. Whereas the cerebellum in GLT‐1‐deficient mice had normal levels of glutamate, glutamine, and 13C labeling of metabolites, glutamate level was decreased but labeling from 1‐13C glucose was unchanged in the neocortex. The contribution from pyruvate carboxylation toward labeling of these metabolites was unchanged. Labeling from 1,2‐13C acetate, originating in astrocytes, was decreased in glutamate and glutamine in the neocortex indicating reduced mitochondrial metabolism in astrocytes. The decreased amount of glutamate in the cortex indicates that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the cortex. Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT‐1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we used 1‐13Cglucose and 1,2‐13Cacetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the neocortex. Glutamate is the major excitatory neurotransmitter, and is inactivated by uptake via GLT‐1 (EAAT2) and GLAST (EAAT1) transporters, while axon terminals in the neocortex only express GLT‐1. To evaluate the role of GLT‐1 in glutamate homeostasis, we used 1‐13Cglucose and 1,2‐13Cacetate injection and NMR spectroscopy. The results indicate that glutamine transport into neurons is not sufficient to replenish glutamate lost because of neurotransmission and that GLT‐1 plays a role in glutamate homeostasis in the neocortex.