1. Calcium-induced transient inactivation of NMDA receptor (NMDAR) channels was studied in cultured rat hippocampal and cerebellar granule neurones using patch-clamp techniques and confocal scanning microscopy. 2. During whole-cell recordings, in the presence of 2 mM external Ca2+, conditioning (2-20 s) pulses of NMDA (20-100 microM) caused a transient decrease in NMDA responses. Recovery developed in two phases with time constants of 0.6 and 40 s. The slow phase of the recovery could be prevented either by strong intracellular Ca2+ ([Ca2+]i) buffering with 30 mM BAPTA or by using Ca(2+)-free extracellular solution. 3. Simultaneous measurement of currents and Ca(2+)-dependent fluorescence revealed a close correlation between the time constants of [Ca2+]i decay and the slow component of NMDA-activated test current recovery. 4. During prolonged recordings, the transient inactivation was not related to irreversible NMDA-activated current run-down. After 25 min of recording with ATP-free intracellular solution, NMDA-activated currents in hippocampal neurones irreversibly decreased by 49 +/- 5% while inactivation decreased by 8% (n = 9). Calyculin A and FK-506 (phosphatase inhibitors) significantly delayed run-down but did not modulate the transient inactivation. 5. In cerebellar granule cells that did not show run-down (4 mM MgATP in the pipette) the percentage of transient inactivation strongly decreased during 25 min of recording (from 28 +/- 6 to 7 +/- 5%, n = 15). 6. In cell-attached recordings (5 microM NMDA in the pipette), elevation of [Ca2+]i (application of 100 microM NMDA to the soma) caused a reversible reduction of single NMDAR channel open probability (NPo) due to a decrease in the frequency of channel opening. 7. In inside-out patches, application of Ca2+ to the cytoplasmic side of the membrane caused a rapid and reversible decrease in NPo (13 out of 29 patches). In the absence of run-down, the ability of Ca2+ to transiently inhibit NMDAR channel activity disappeared after 3-5 min of recording. 8. These results indicate that Ca(2+)-induced transient inactivation of NMDAR currents develops independently from the run-down and suggest that a diffusible Ca2+ -dependent factor mediates NMDAR channel inactivation.