High affinity uptake of glutamate plays a major role in the termination of excitatory neurotransmission. Identification of the ramifications of transporter function is essential to understand the diseases in which defective excitatory amino acid transporters (EAAT) have been implicated. In this work we incubated Guinea pig cortical tissue slices with [3-13C]pyruvate and major currently available glutamate uptake inhibitors and studied the resultant metabolic sequelae by 13C and 1H NMR spectroscopy using a multivariate statistical approach. Perturbation of glutamate uptake produced significant effects on metabolic flux through the Krebs cycle, and on glutamate/glutamine cycling rates, with this effect accounting for 76% of the variation in the total data set. The effects of all inhibitors were separable from each other along three major principal components. The competitive inhibitor l-CCG III ((2S,1′S,2′R)-2-carboxycyclopropyl)glycine) differed most from the other inhibitors, showing negative weightings on both the first and second principal components, whereas the EAAT2-specific inhibitor dihydrokainate (DHK) showed metabolic patterns similar to that of anti-endo-3,4-methanopyrolidine dicarboxylate but separate from those of dl-threo-β-benzyloxyaspartate (TBOA) and l-trans-pyrrolidine-2,4-dicarboxylate (l-tPDC). This indicates that different inhibition mechanisms or different colocalisation of the separate transporter subtypes with glutamate receptors can produce significantly different metabolic and functional outcomes for the brain. © 2006 Wiley-Liss, Inc.