Changes in extracellular free calcium concentration ([Ca2+]o) were measured with ion-selective microelectrodes in the dentate gyrus of the rat hippocampal slice preparation. Repetitive stimulation (20 Hz/10 s) of the perforant path or mossy fibers, or alternatively, iontophoresis of the excitatory amino acids N-methyl-D-aspartate (NMDA) or quisqualate (Quis) elicited decreases in [Ca2+]o (delta Ca2+) which were unequally distributed along the axis of the granule cells. Laminar profiles of the [Ca2+]o changes revealed marked differences between stimulus- and amino acid-induced responses. The delta Ca2+ induced by either anti- or orthodromic stimulation were relatively small (less than 0.15 mM) and were found to be maximal at the cell body layer (stratum granulosum). In contrast, the excitatory amino acids NMDA and Quis evoked large delta Ca2+ (greater than 1.2 mM) which were maximal at dendritic sites, 100 microns away from the cell body layer in the inner stratum moleculare. The effect of NMDA was reversibly blocked by 2-amino-5-phosphonovaleric acid without antagonism of the synaptic responses or the stimulus-induced changes in [Ca2+]o. Therefore, under normal conditions, NMDA receptors appear not to participate in synaptically induced delta Ca2+ in the dentate gyrus.