Calcium-dependent inactivation of NMDA channels was examined on cultured rat hippocampal neurons using whole-cell voltage-clamp and cell-attached single-channel recording. An ATP regeneration solution was included in the patch pipette to retard current “rundown.” In normal [Ca2+]o (1-2 mM) and 10 microM glycine, macroscopic currents evoked by 15 sec applications of NMDA (10 microM) inactivated slowly following an initial peak. At -50 mV in cells buffered to [Ca2+]i < 10(- 8) M with 10 mM EGTA, the inactivation time constant (tau inact) was approximately 5 sec. Inactivation did not occur at membrane potentials of +40 mV and was absent at [Ca2+]o < or = 0.2 mM, suggesting that inactivation resulted from transmembrane calcium influx. The percentage inactivation and tau inact were dependent on [Ca2+]o. The tau inact was also longer with BAPTA in the whole-cell pipette compared to EGTA, suggesting that tau inact reflects primarily the rate of accumulation of intracellular calcium. Inactivation was incomplete, reaching a steady state level of 40–50% of the peak current. At steady state, block of open NMDA channels with MK-801 ((+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,10-imine) completely blocked subsequent responses to NMDA, suggesting that “inactivated” channels can reopen at steady state. Inactivation was fully reversible in the presence of ATP but was not blocked by inhibiting phosphatases or proteases. In cell- attached patches, transient increases in [Ca2+]i following cell depolarization also resulted in inactivation of NMDA channels without altering the single-channel conductance. This suggests that Ca(2+)- dependent inactivation occurs in intact cells and can be triggered by calcium entry through nearby voltage-gated calcium channels, although calcium entry through NMDA channels was more effective. We suggest that [Ca2+]i transients induce NMDA channel inactivation by binding to either the channel or a nearby regulatory protein to alter channel gating. This mechanism may play a role in downregulation of postsynaptic calcium entry during sustained synaptic activity.