¹³C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-¹³C]/[1,6-¹³C₂]glucose as labeled substrate have consistently found a lower enrichment (~25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using ¹H-[¹³C]-NMR spectroscopy together with intravenous infusion of [U-¹³C₅]glutamine for 3, 15, 30, and 60 minutes in mice. ¹³C labeling of brain glutamine was found to be saturated at plasma glutamine levels > 1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the ¹³C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, V_Gln(in), 0.036 ± 0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (~0.6 mmol/L), V_Gln(in) would be ~0.010 μmol/g per minute, which corresponds to ~6% of the glutamine synthesis rate and rises to ~11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ~20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-¹³C]glucose.