Metabolic modeling of 13C NMR spectroscopy (13C MRS) data using two-compartment neuronal-glial models enabled non-invasive measurements of the glutamate-glutamine cycle rate (VNT) in the brain in vivo. However, the reliability of such two-compartment metabolic modeling has not been examined thoroughly. This study uses Monte-Carlo simulations to investigate the reliability of metabolic modeling of 13C positional enrichment time courses measured in brain amino acids such as glutamate and glutamine during [1-13C]- or [1,6-13C2]glucose infusion. Results show that the determination of VNT is not very precise under experimental conditions typical of in vivo NMR studies, whereas the neuronal TCA cycle rate VTCA(N) is determined with a much higher precision. Consistent with these results, simulated 13C positional enrichment curves for glutamate and glutamine are much more sensitive to the value of VTCA(N) than to the value of VNT. We conclude that the determination of the glutamate-glutamine cycle rate VNT using 13C MRS is relatively unreliable when fitting 13C positional enrichment curves obtained during [1-13C] or [1,6-13C2]glucose infusion. Further developments are needed to improve the determination of VNT, for example using additional information from 13C-13C isotopomers and/or using glial specific substrates such as [2-13C]acetate. © 2007 Wiley-Liss, Inc.