The aim of this study was to compare the diffusion characteristic of lactate and alanine in a brain tumor model to that of normal brain metabolites known to be mainly intracellular such as N-acetylaspartate or creatine. The diffusion of 13 C-labeled metabolites was measured in vivo with localized NMR spectroscopy at 9.4T (400MHz) using a previously described localization and editing pulse sequence known as ACED-STEAM (‘adiabatic carbon editing and decoupling’). 13 C-labeled glucose was administered and the apparent diffusion coefficients of the glycolytic products, { 1 H– 13 C}-lactate and { 1 H– 13 C}-alanine, were determined in rat intracerebral 9L glioma. To obtain insights into { 1 H– 13 C}-lactate compartmentation (intra- versus extracellular), the pulse sequence used very large diffusion weighting (50ms/μm 2 ). Multi-exponential diffusion attenuation of the lactate metabolite signals was observed. The persistence of a lactate signal at very large diffusion weighting provided direct experimental evidence of significant intracellular lactate concentration. To investigate the spatial distribution of lactate and other metabolites, 1 H spectroscopic images were also acquired. Lactate and choline-containing compounds were consistently elevated in tumor tissue, but not in necrotic regions and surrounding normal-appearing brain. Overall, these findings suggest that lactate is mainly associated with tumor tissue and that within the time-frame of these experiments at least some of the glycolytic product ([ 13 C] lactate) originates from an intracellular compartment.