Repetitive stimulation of glutamate (glu) receptors elicits increasingly smaller ionic currents in hippocampal neurons. To investigate mechanisms underlying this phenomenon, voltage clamp whole-cell currents evoked by glu (100 microM) were recorded from hippocampal neurons in culture. These currents were primarily carried by N-methyl-D-aspartate-receptor (NMDA-R) channels, as shown by the voltage-dependent sensitivity to extracellular Mg2+ blockade, and inhibition by the specific antagonist MK-801. In the presence of 2.2 mM extracellular Ca2+ ([Ca2+]e), repetitive glu applications (15 episodes of 4 s/min) elicited progressively smaller currents that stabilized at 45% of their initial peak value. Replacement of [Ca2+]e by Ba2+ produced similar effects. This phenomenon, defined as interepisode inactivation, was exacerbated by elevating [Ca2+]e to 11 mM, attenuated by reducing [Ca2+]e to 0.22 mM, and further diminished by shortening the length of the glu pulse to 2 s. Current decay exhibited during individual stimuli, or intraepisode inactivation, was dependent on [Ca2+]e yet remained stable during repetitive stimulation. We conclude that interepisode and intraepisode inactivations of NMDA-R currents are the expression of two distinct processes triggered by Ca2+. These modalities of inactivation may arise from Ca2+ binding either to the receptor or to closely associated regulatory proteins.