1. Agonist-independent (inactivation) and agonist-induced (desensitization) refractory states of N-methyl-D-aspartate (NMDA) receptors were studied on cultured rat hippocampal neurones using whole-cell and inside-out patch-clamp techniques and a fast perfusion system. 2. Shortly after whole-cell formation, application of 100 microM NMDA in the presence of 10 microM glycine and 0.2 mM [Ca2+]o induced membrane currents that desensitized by 23%. Repeated application of NMDA at 30 s intervals resulted in a progressive increase in the degree and rate of onset of NMDA receptor desensitization. 3. Test responses to NMDA recorded in the presence of 0.2 mM [Ca2+]o were reversibly inactivated by 60% following a train of ten 1 s applications of NMDA delivered at 0.5 Hz in the presence of 2 mM [Ca2+]o; similar results were obtained with 2 mM [Sr2+]o and 2 mM [Ba2+]o. In the presence of Ca2+ or Sr2+, desensitization during the train of responses to NMDA increased by 14 and 19% respectively, while with Ba2+ there was no increase in desensitization. 4. In the presence of 0.2 mM [Ca2+]o at a holding potential of -60 mV, or in the presence of 2 mM [Ca2+]o at a holding potential of +50 mV, a train of ten applications of NMDA failed to induce either inactivation or an increase in desensitization of test responses to NMDA. These results suggest an important role for [Ca2+]o in the induction of both inactivation and desensitization of NMDA receptors. 5. Increasing the intracellular calcium concentration, [Ca2+]i, via repeated activation of voltage-gated Ca2+ channels, resulted in a reversible inactivation of test responses to NMDA by 35% but failed to increase desensitization. In neurones dialysed with intracellular solution containing 2.5 mM Ca2+ NMDA receptor desensitization was similar to that in neurones dialysed with 10 nM Ca2+. 6. Block of NMDA receptor-channels by 2 mM [Mg2+]o during the train application of NMDA prevented the induction of both inactivation and desensitization. In contrast 3 mM [Mg2+]i was ineffective. 7. The magnitude of both inactivation and desensitization of NMDA receptors was not affected by intracellular dialysis of ATP, the non-hydrolysable ATP analogue 5'-adenylylimido-diphosphate (AMP-PNP), different Ca2+ chelators (EGTA or BAPTA), the Ca(2+)-activated protease inhibitor (leupeptin), dithiothreitol, or the phosphatase inhibitors (okadaic acid and a calcineurin inhibitor). 8. Application of 2.5 mM Ca2+ to the cytoplasmic side of inside-out patches induced inactivation of NMDA responses similar in magnitude to the inactivation seen in whole-cell recording.(ABSTRACT TRUNCATED AT 400 WORDS)