(13)C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-(13)C]/[1,6-(13)C2]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 (1)H-[(13)C]-NMR spectroscopy together with intravenous infusion of [U-(13)C5]glutamine for 3, 15, 30, and 60 minutes in mice. (13)C 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 (13)C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(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), VGln(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-(13)C]glucose.