Glial cells are currently viewed as active partners of neurons in synapse formation. The close proximity of astrocytes to the synaptic cleft suggests that these cells might be potential targets for neuronal-released molecules although this issue has been less addressed. Here, we evaluated the role of the excitatory neurotransmitter, glutamate, in astrocyte differentiation. We recently demonstrated that cortical neurons activate the gene promoter of the astrocyte maturation marker, GFAP (glial fibrillary acidic protein) of cerebral cortex astrocytes by inducing TGF-beta1 (transforming growth factor beta 1) secretion in vitro. To access the effect of glutamate on GFAP gene, we used transgenic mice bearing the beta-Galactosidase (beta-Gal) reporter gene under the regulation of the GFAP gene promoter. We report that 100 muM glutamate activates the GFAP gene promoter of astrocytes from cerebral cortex revealed by a significant increase in the number of beta-Gal positive astrocytes. Neutralizing antibodies against TGF-beta completely prevented glutamate and neuronal-induction of GFAP gene, thus indicating that this event is mediated by TGF-beta1. Further, induction of GFAP gene in response to glutamate was followed by nuclear translocation of the Smad transcription factor, a hallmark of TGF-beta1 pathway activation. The antagonist of the metabotropic glutamate receptor, MCPG, inhibited neuronal effect suggesting that neuronal activation of GFAP gene promoter involves glutamate metabotropic receptors. MAPK (PD98059) and PI3K (LY294002) inhibitors fully prevented activation of GFAP gene promoter by all treatments. Surprisingly, these inhibitors also abrogated TGF-beta1 direct action on GFAP gene although they did not inhibit Smad-2 phosphorylation, suggesting that TGF-beta1-induced GFAP gene activation might involve cooperation between the canonical and non-canonical TGF-beta pathways. Together, our results suggest that glial metabotropic glutamate 2/3 receptor activation by neurons induces TGF-beta1 secretion, leading to GFAP gene activation and astrocyte differentiation and involves Smad and MAPK/PI3K pathways. Our work provides evidence that astrocytes surrounding synapses are target of neuronal activity and might shed light into the role of glial cells into neurological disorders associated with glutamate neurotoxicity.