Glutamate triggers an acute stimulation of the glucose transporter GLUT1 in cultured astrocytes, a phenomenon thought to facilitate energy delivery to active areas in the brain. Here we have explored the cell signaling mechanisms involved in this response. Half-stimulation of GLUT1 occurred at low micromolar glutamate, thus within the physiological range estimated in brain interstitium. The effect was mimicked by D-aspartate and inhibited by L-threo-beta-benzyloxyaspartate or Na+ replacement with NMDG+, showing the participation of the Na+-glutamate co-transporter. AMPA and the mGLURI agonist DHPG had no effect. The stimulation of GLUT1 was fully inhibited by ouabain, but independent activation of the Na+/K+ ATPase pump with gramicidin did not affect glucose transport. Simultaneous with the Na+ rise, glutamate and D-aspartate triggered a Ca2+signal, whose inhibition with BAPTA prevented the stimulation of GLUT1. However, an isolated Ca2+ signal, triggered with endothelin 1, ATP or DHPG, did not affect glucose transport. The stimulation of GLUT1 could finally be mimicked by simultaneous induction of Na+ and Ca2+ signals. The requirement for both cations in the stimulation of the astrocytic glucose transporter, may help to explain how glucose metabolism in the brain is strongly activated by glutamate, but not by GABA or by inter-astrocytic signaling. © 2007 Wiley-Liss, Inc.