The ability of excitatory amino acids to induce increases in the intracellular Ca2+ concentration ([Ca2+]i) of cerebellar Purkinje cells was examined by digital fluorescence ratio imaging of voltage-clamped Purkinje cells dialyzed with the Ca2+ indicator fura-2. Purkinje cells responded with large inward currents accompanied by increases in dendritic [Ca2+]i when challenged with the excitatory amino acid agonists glutamate and quisqualate. The rise in [Ca2+]i was transient and reached peak values of several hundred nanomolar. The response subsisted in the absence of extracellular Ca2+, a condition that eliminates Ca2+ entry through voltage-gated Ca2+ channels, indicating that Ca2+ arose in large part from an intracellular compartment. In support of this hypothesis, only the first agonist application elicited a [Ca2+]i increase in slices maintained in Ca(2+)-free medium, as expected if the intracellular stores become depleted. These results indicate that metabotropic glutamate receptors are functional in Purkinje cells and point to glutamate as a possible modulator of [Ca2+]i in these neurons.