ATP can be significantly released following various brain insults and activates the extracellular signal-regulated protein kinase (ERK) pathway in astrocytes. Glutamate transporter-1 (GLT1) is the major forebrain astroglial glutamate transporter and its expression is stimulated also via ERK1/2 phosphorylation. We thus hypothesized that extracellular ATP could be a signal to GLT1 modulation in hippocampal slices obtained from rat. We indeed observed by western blot analysis that, after 1 mM ATP exposure, GLT1 expression, but not the glutamate–aspartate transporter, was enhanced. At the same time, high ATP induced significant rates of cell death in piramidal and granule cell layers, as shown by propidium iodide uptake, and increased glutamate uptake through GLT1 transporter. Also using confocal laser-scanning microscopy, we observed that ATP induced a vigorous and extensive GLT1-labeling on glial fibrillary acidic protein-positive cells. This stimulation was abolished by purine/pyrimidine nucleotide receptor antagonists and by MEK1/2 inhibitor. The present study demonstrates a novel mechanism of GLT1 regulation by extracellular ATP, reinforcing the evidence of cross talk between glutamatergic and purinergic systems. © 2007 Wiley-Liss, Inc.