The glutamate receptors (GluRs) are key receptors for modulatory synaptic events in the central nervous system. It has been reported that glutamate increases the intracellular Ca2⁢ concentration ([Ca2⁢]i) and induces cytotoxicity. In the present study, we investigated whether the glutamate-induced [Ca2⁢]i increase was associated with the activation of ionotropic (iGluR) and metabotropic GluRs (mGluR) in substantia gelatinosa neurons, using spinal cord slice of juvenile rats (10∼21 day). [Ca2⁢]i was measured using conventional imaging techniques, which was combined with whole-cell patch clamp recording by incorporating fura-2 in the patch pipette. At physiological concentration of extracellular Ca2⁢, the inward current and [Ca2⁢]i increase were induced by membrane depolarization and application of glutamate. Dose-response relationship with glutamate was observed in both Ca2⁢ signal and inward current. The glutamate-induced [Ca2⁢]i increase at holding potential of ⁣70 mV was blocked by CNQX, an AMPA receptor blocker, but not by AP-5, a NMDA receptor blocker. The glutamate-induced [Ca2⁢]i increase in Ca2⁢ free condition was not affected by iGluR blockers. A selective mGluR (group I) agonist, RS-3,5-dihydroxyphenylglycine (DHPG), induced [Ca2⁢]i increase at holding potential of ⁣70 mV in SG neurons. These findings suggest that the glutamate-induced [Ca2⁢]i increase is associated with AMPA-sensitive iGluR and group I mGluR in SG neurons of rats.