Currently, the majority of studies concerning the regulation of glutamate transporters are performed on mixed cultures of cortical astrocytes. However, one should certainly predict that astrocytes found in the grey or white matter of the central nervous system show distinct phenotypical specifications. While transmission of excitatory glutamate inputs is certainly predominant in synapses within the grey matter, recent studies evidenced vesicular glutamate release from axons in white matter area. Such observations raise questions regarding possible differences in glial glutamate uptake systems in these regions. Therefore, we herein present a detailed characterization of cultures of grey and white matter astrocytes in terms of cell phenotype and expression of intermediate filaments as well as expression and function of the major glutamate transporters GLAST and GLT-1. These populations of astrocytes showed clearly distinct morphologies, respectively adopting fibrous or stellate shapes. Indeed, white matter astrocytes showed high densities of the intermediate filaments GFAP, vimentin and nestin. The glutamate transporters GLAST and GLT-1 as well as glutamine synthetase were found to be expressed at higher levels in white matter as compared to grey matter astrocytes. Consistently, aspartate uptake capacity was 3–4 fold higher in white matter cells, and the use of specific inhibitors revealed the substantial activity of GLT-1, contrasting with grey matter cells where this transport was poorly functional. Noticeable differences were also observed in the properties of these cultures when exposed to chemical stimuli known to favor the maturation/activation of astrocytes and mimicking a gliotic environment. All together, these observations highlight typical features of white matter astrocytes which appear constitutively adapted to face excitotoxic insults.