In the present work, diffusion-weighted (DW)-NMR spectroscopy of glutamate was performed during a 13 C-labeled glucose infusion in monkey brain (six experiments). It is shown that glutamate 13 C labeling occurs significantly faster at higher diffusion weightings-slightly for glutamate in position C4, and more markedly for glutamate in position C3. This demonstrates the existence of different diffusion compartments for glutamate, associated with different metabolic rates. Metabolic modeling of 13 C enrichment time-courses suggests that these compartments might be gray and white matter, each having a specific oxidative metabolism rate possibly paralleled by a specific glutamate diffusion coefficient. Given its sensitivity to the molecular diffusion of metabolites, diffusion-weighted (DW)-NMR spectroscopy is generally accepted to be a valuable tool for probing various cellular compartments in vivo (for review, see Ref. 1). Indeed, the apparent diffusion coefficient (ADC) as measured by DW-NMR potentially depends on a wide variety of parameters that might be characteristic of different compartments, such as intracellular viscosity and tortuosity, restriction by or exchange through membranes, vesicle diffusions, or even cytoplasmic streaming. This expected sensitivity to cellular and subcellular properties has motivated various studies aimed at measuring metabolite ADC in the normal brain (2-10) or under perturbative conditions such as ischemia (11-16), excitotoxic injury (13), tumor (16), or variation of the anesthesia level (17). However, although there is little doubt about the actual existence in the brain of compartments with different diffusion properties, they have not been clearly identified, and the extent to which DW spectroscopy effectively allows them to be probed in vivo remains to be explored experimentally.A comparison of ADC values measured in the monkey brain and the rat brain led us to hypothesize that the molecular diffusion of metabolites, and particularly glutamate, could occur faster in white matter (WM) as compared to gray matter (GM) (6). A recent study in which the ADC was measured for NAA, creatine, and choline in different regions of the human brain seemed to confirm this trend (8), although the origin of the GM-WM discrepancy is unclear. We recently proposed that the ADC may also reflect subcellular compartmentation (in the cytoplasm and the different organelles [17]). In the end, regardless of the level at which diffusion properties vary, diffusion compartments are likely to match anatomical/functional compartments. It is therefore legitimate to expect some metabolic difference associated with these various diffusion compartments, which might give new insights into cellular compartmentation in the brain.13 C-NMR spectroscopy is a powerful tool for assessing oxidative metabolism in vivo. The measurement relies on the dynamic detection, during a 13 C-labeled glucose infusion, of 13 C incorporation into the concentrated aminoacid glutamate (for review see Refs. 18 and 19). Combining oxidativ...