The N-methyl-D-aspartate (NMDA) receptor is unique among the ligand-gated ion channels, in that the gating process requires the binding of two independent coagonists, glutamate and glycine. Receptor binding experiments have suggested that the coagonist recognition sites interact with one another in an allosteric manner, and previous work in this laboratory has provided additional functional support in favor of an allosteric coupling; the affinity of glutamate for its recognition site was reduced when a partial agonist, (+)-HA-966, occupied the glycine site, compared with the affinity when glycine itself was bound to the receptor. The present experiments have taken these observations a step further and compare the effects of several glycine site ligands with different affinities and intrinsic activities (determined from equilibrium concentration-response curves) on glutamate off-rate. Thus, the dissociation rate for the decay of glutamate-activated membrane currents in voltage-clamped rat cortical neurons was fastest (160 +/- 28 msec) in the presence of saturating concentrations of (+)-HA-966 and progressively slower in the presence of D-cycloserine (258 +/- 27 msec), aminocyclopropanecarboxylic acid (330 +/- 21 msec), L-alanine (375 +/- 28 msec), and glycine (502 +/- 42 msec). We have also measured the affinities and intrinsic activities of several NMDA receptor ligands and report that a reciprocal interaction exists, such that the off-rate of glycine is influenced by the properties of the agonist occupying the glutamate coagonist site. Thus, the time constant for current decay after a brief exposure to glycine was fastest in the presence of a saturating concentration of cis-2,3-piperidinedicarboxylic acid (449 +/- 26 msec) and progressively slower in the presence of quinolinate (689 +/- 73 msec), NMDA (721 +/- 36 msec), and L-glutamate (1260 +/- 36 msec). The data suggested that the extent of the modulation of one site by the other is related to the intrinsic activity of the agonist, rather than its affinity. Specifically, we suggest that a partial agonist occupying one of the agonist recognition sites produces a conformational change that results in an accelerated off-rate for coagonist dissociation from the receptor; the lower the intrinsic activity, the greater is the effect on coagonist off-rate.