We report the results of microdialysis experiments investigating the NMDA-induced release of intracellular Ca2+ in different brain regions. Microdialysis probes were implanted stereotaxically into the striatum, thalamus and hippocampus dentate gyrus (DG) of adult rats. Dialysates were analysed for alterations in the concentration of ionized Ca2+ in an initially calcium-free medium and for changes in 45Ca efflux from the pre-labelled endogenous Ca2+ pools. The application of 5 mM of NMDA to the dialysis medium for 20 min in the striatum, resulted in increases in Ca2+ and 45Ca concentrations by 25% and 35% respectively. After NMDA perfusion in the hippocampus DG and in the thalamus, decreases in the Ca2+ concentration to 65.6% and 38.6% of the basal level respectively, were accompanied by increases in 45Ca efflux, exceeding 1,500% of the basal level in the hippocampus. Cell swelling, and the corresponding reduction of the extracellular space volume was insufficient to explain the huge increase in 45Ca efflux. Thus, our experiments demonstrated that in vivo in the rat hippocampus DG, NMDA induces the release of 45Ca to the extracellular space from unidentified intracellular calcium stores.