In this study, cellular distribution and activity of glutamate and γ-aminobutyric acid (GABA) transport as well as oxoglutarate transport across brain mitochondrial membranes were investigated. A goal was to establish cell-type-specific expression of key transporters and enzymes involved in neurotransmitter metabolism in order to estimate neurotransmitter and metabolite traffic between neurons and astrocytes. Two methods were used to isolate brain mitochondria. One method excludes synaptosomes and the organelles may therefore be enriched in astrocytic mitochondria. The other method isolates mitochondria derived from all regions of the brain. Immunological and enzymatic methods were used to measure enzymes and carriers in the different preparations, in addition to studying transport kinetics. Immunohistochemistry was also employed using brain slices to confirm cell type specificity of enzymes and carriers. The data suggest that the aspartate/glutamate carriers (AGC) are expressed predominantly in neurons, not astrocytes, and that one of two glutamate/hydroxyl carriers is expressed predominantly in astrocytes. The GABA carrier and the oxoglutarate carrier appear to be equally distributed in astrocytes and neurons. As expected, pyruvate carboxylase and branched-chain aminotransferase were predominantly astrocytic. Insofar as the aspartate/glutamate exchange carriers are required for the malate/aspartate shuttle and for reoxidation of cytosolic NADH, the data suggest a compartmentation of glucose metabolism in which astrocytes catalyze glycolytic conversion of glucose to lactate, whereas neurons are capable of oxidizing both lactate and glucose to CO2 + H2O. © 2007 Wiley-Liss, Inc.