The effects of agonist concentration, transmembrane voltage and calcium on the glycine-independent desensitization of N-methyl-D-aspartate receptors were examined in voltage-clamped postnatal rat hippocampal neurons. In the presence of 1 micron glycine and 1 mM calcium, both the time-course and degree of N-methyl-D-aspartate desensitization were dependent on agonist concentration. Transmembrane voltage influenced both the rate and degree of desensitization with a more rapid rate and greater degree at negative holding potentials. Both the time-course and the degree of desensitization were influenced by extracellular calcium in a dose-dependent manner. N-Methyl-D-aspartate desensitization is not augmented by activation of voltage-gated calcium currents and is more pronounced in the presence of intracellular calcium chelators, suggesting that the site of action of calcium in promoting desensitization is extracellular. Although a physiological role for N-methyl-D-aspartate desensitization has not been demonstrated, the process is observed over a range of agonist concentrations, is prominent near resting membrane potential and is regulated by physiologic concentrations of calcium. Thus desensitization could be an important neuroprotective mechanism during periods of prolonged glutamate exposure.