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Abstract Monitoring the metabolism of 13C-labelled substrates by biological tissues allows both the rate of metabolism and the relative importance of metabolic pathways to be determined. In this study high-resolution magic-angle spinning (HRMAS) 13C NMR spectroscopy is assessed as a technique for determining the labelling of metabolites in brain slices. Freshly prepared rat brain slices were superfused in isotonic salt solution containing [1-13C] glucose. HRMAS 1H and 13C NMR spectra were acquired of the slices (~10 mg) at 3°C. Using 1H NMR spectroscopy it was demonstrated that the concentration of key metabolites indicative of metabolic degradation, including N-acetyl aspartate and lactate, did not change significantly across the ~11 h time period required for 13C NMR spectra. The approach produced high-resolution spectra of intact tissue with the labelling patterns of tissues being indicative of both labelling via pyruvate dehydrogenase found in both neuronal and glial cells, and pyruvate carboxylase, found only within glial cells. This approach is a versatile tool for monitoring the compartmentation of metabolites directly, and will also allow the investigation of aqueous and lipid metabolites simultaneously. [ABSTRACT FROM AUTHOR]