The Ca2+ sensitive fluorescent probe, fura-2 has been used to monitor cytosolic free calcium levels in mature primary cultures of cerebellar granule cells during exposure to L-glutamate and other excitatory amino acids: quisqualate (QA) kainate (KA) and N-methyl-D-aspartate (NMDA). Glutamate at micromolar concentrations produced a prompt and dose-related increase in the intracellular concentration of free Ca2+, ([Ca2+]i), whereas QA, KA and NMDA had no effect. This increase was also seen in the absence of extracellular Ca2+, suggesting that L-glutamate promotes mobilization of Ca2+ from intracellular stores. In the presence of extracellular calcium, the elevation of [Ca2+]i was, in part, mediated by an increase in the plasma membrane permeability to Ca2+. This Ca2+ influx was not affected by the Ca2+-channel antagonist l-Verapamil. However, L-Verapamil did block the increase in [Ca2+]i seen after depolarization of the cells with potassium. The Ca2+ response elicited by glutamate was partially blocked by the excitatory amino acid antagonist glutamate diethyl ester (GDEE). Furthermore, glutamate stimulated the formation of inositol mono-, bis-, tris- and tetrakisphosphates (IP1, IP2, IP3, and IP4) suggesting a role for these compounds for the increase in [Ca2+]i.