In neural cells, Na+/Ca2+ exchanger (NCX) participates in Ca2+ recycling across mitochondrial membranes, thus contributing to shape Ca2+ responses. NCX exchanger isoform proteins, NCX1-3, are widely distributed in mammalian brain, where they localize to neuronal, glial and endothelial cells, but anatomical data on their mitochondrial expression are scanty. In the present work, mitochondrial localization of NCX1-3 was investigated in rat neocortex and hippocampus by means of western blotting analysis and in situ electron microscopy immunocytochemistry. Results showed that a conspicuous population of neuronal and astrocytic mitochondria express NCX1-3, with distinct isoforms exhibiting differential patterns of mitochondrial expression. In neurons, percentages of NCXs-labelled mitochondria varied significantly between diverse subcellular regions: the majority of NCXs-expressing mitochondria were found in dendrites, often located beneath the plasmalemma and near postsynaptic sites. In astrocytes, most NCXs-labelled mitochondria were situated close to the cellular surface. Present quantitative and qualitative immunocytochemical data suggest that all NCX isoforms contribute to mitochondrial Ca2+ homeostasis in neurons and glial cells in vivo, and that NCXs may be particularly involved in handling Ca2+ in dendritic, subplasmalemmal mitochondria, thus emphasizing the role of mitochondrial NCX1-3 in shaping postsynaptic calcium transients.