1. Ionic currents and the cytosolic free calcium concentration ([Ca2+]i) were recorded in rat hippocampal neurons in culture using the whole-cell configuration of the patch-clamp technique and confocal laser scanning microscopy with the fluorescent Ca2+ indicator Fluo-3 or dual-emission microspectrofluorimetry with the fluorescent Ca2+ indicator Indo-1. The excitatory amino acids, kainate and N-methyl-D-aspartate (NMDA), were repeatedly applied to the neurons using either a fast perfusion system or pressure-ejection from micropipettes. 2. Conditioning (1-10 s) applications of NMDA induced desensitization of NMDA currents. Recovery from desensitization, estimated from analysis of the amplitudes of short (20-50 ms) test NMDA currents, was double exponential. The time constant of the first phase was < 2 s and for the second phase it was in the range 10-50 s. 3. Conditioning applications of kainate decreased the amplitude of NMDA currents. Recovery of NMDA currents from kainate-induced inactivation was slow and could be fitted with a single exponential. The time constant of recovery was in the range 10-50 s and increased with prolongation of the conditioning pulse of kainate. 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM) prevented kainate-induced inactivation of NMDA currents. 4. Depolarizing voltage pulses (1-10 s) also induced an inactivation of NMDA currents with a slow recovery. The time course of the recovery increased with prolongation of depolarizing pulses and with an elevation of external calcium. Cadmium, a blocker of voltage-gated channels, prevented development of the depolarization-induced inactivation of NMDA currents. 5. Simultaneous recording of ionic currents and fluorescence of Ca(2+)-sensitive dyes showed that application of kainate, NMDA, or depolarizing pulses resulted in a rise of [Ca2+]i. Cadmium (100 microM) reversibly blocked [Ca2+]i transients induced by depolarizing pulses without modification of kainate-induced rise in fluorescence intensity. 6. For equal inward currents the elevation of [Ca2+]i was approximately 3.5-fold higher for applications of NMDA than for kainate. 7. Strong buffering of [Ca2+]i prevented the inactivation of NMDA currents induced by kainate or by depolarization. 8. Our results suggest that in the hippocampal neurons kainate produces inactivation of NMDA currents via an elevation of [Ca2+]i.