We have previously demonstrated that rat cerebellar Type-1 astrocytes express a very active genistein sensitive Na+/Ca2+ exchanger, which accounts for most of the total plasma membrane Ca2+ fluxes and for the clearance of Ca2þ i loads induced by physiological agonists. In this work, we have explored the mechanism by which the reverse Na+/Ca2+ exchange is involved in agonist-induced Ca2+ signaling in rat cerebellar astrocytes. Microspectrofluorometric measurements of Ca2þ i with Fluo-3 demonstrate that the Ca2þ i signals associated long (> 20 s) periods of reverse operation of the Na+/Ca2+ exchange are amplified by a mechanism compatible with calcium-calcium release, while those associated with short (< 20 s) pulses are not amplified. This was confirmed by pharmacological experiments using ryanodine receptors agonist (4-chloro-m-cresol) and the endoplasmic reticulum ATPase inhibitor (thapsigargin). Confocal microscopy demonstrates a high co-localization of immunofluorescent labeled Na+/Ca2+ exchanger and RyRs. Low (< 50 lmol/L) or high (> 500 lmol/L) concentrations of L-glutamate (L-Glu) or L-aspartate causes a rise in Ca2þ i which is completely blocked by the Na+/Ca2+ exchange inhibitors KB-R7943 and SEA0400. The most important novel finding presented in this work is that L-Glu activates the reverse mode of the Na+/Ca2+ exchange by inducing Na+ entry through the electrogenic Na+–Glu-co-transporter and not through the ionophoric L-Glu receptors, as confirmed by pharmacological experiments with specific blockers of the ionophoric L-Glu receptors and the electrogenic Glu transporter.