Localized, water‐suppressed ¹H‐[¹³C]‐NMR spectroscopy was used to detect ¹³C‐label accumulation in cerebral metabolites following the intravenous infusion of [1,6‐¹³C₂]‐glucose (Glc). The ¹H‐[¹³C]‐NMR method, based on adiabatic RF pulses, 3D image‐selected in vivo spectroscopy (ISIS) localization, and optimal shimming, yielded high‐quality ¹H‐[¹³C]‐NMR spectra with optimal NMR sensitivity. As a result, the ¹³C labeling of [4‐¹³C]‐glutamate (Glu) and [4‐¹³C]‐glutamine (Gln) could be detected from relatively small volumes (100 μL) with a high temporal resolution. The formation of [n‐¹³C]‐Glu, [n‐¹³C]‐Gln (n = 2 or 3), [2‐¹³C]‐aspartate (Asp), [3‐¹³C]‐Asp, [3‐¹³C]‐alanine (Ala), and [3‐¹³C]‐lactate (Lac) was also observed to be reproducible. The ¹³C‐label incorporation curves of [4‐¹³C]‐Glu and [4‐¹³C]‐Gln provided direct information on metabolic pathways. Using a two‐compartment metabolic model, the tricarboxylic acid (TCA) cycle flux was determined as 0.52 ± 0.04 μmol/min/g, while the glutamatergic neurotransmitter flux equaled 0.25 ± 0.05 μmol/min/g, in good correspondence with previously determined values. Magn Reson Med 49:37–46, 2003. © 2003 Wiley‐Liss, Inc.