The Ν-methyl-D-aspartate (NMDA) receptor channel is an obligatory heterotetramer formed by two GluN1 and two GluN2 subunits. However, the differential contribution of the two different subunits to channel operation is not clear. We found that the apparent affinity of glycine to GluN1 (K gly ∼ 0.6 μM) is much higher than NMDA or glutamate to GluN2 (K NMDA ∼ 36 μM, K glu ∼ 4.8 μM). The binding rate constant (derived from the linear regression of the apparent macroscopic binding rates) of glycine to GluN1 (∼9.8 × 10(6) M(-1) s(-1)), however, is only slightly faster than NMDA to GluN2 (∼4.1 × 10(6) M(-1) s(-1)). Accordingly, the apparent unbinding rates of glycine from activated GluN1 (time constant ∼2 s) are much slower than NMDA from activated GluN2 (time constant ∼70 ms). Moreover, the decay of NMDA currents upon wash-off of both glycine and NMDA seems to follow the course of NMDA rather than glycine unbinding. But if only glycine is washed off, the current decay is much slower, apparently following the course of glycine unbinding. The apparent binding rate of glycine to the fully deactivated channel, in the absence of NMDA, is roughly the same as that measured with co-application of both ligands, whereas the apparent binding rate of NMDA to the fully deactivated channel in the absence of glycine is markedly slower. In this regard, it is interesting that the seventh residue in the highly conserved SYTANLAAF motif (A7) in GluN1 and GluN2 are so close that they may interact with each other to control the dimension of the external pore mouth. Moreover, specific mutations involving A7 in GluN1 but not in GluN2 result in channels showing markedly enhanced affinity to both glycine and NMDA and readily activated by only NMDA, as if the channel is already partially activated. We conclude that GluN2 is most likely directly responsible for the activation gate of the NMDA channel, whereas GluN1 assumes a role of more global control, especially on the gating conformational changes in GluN2. Structurally, this intersubunit regulatory interaction seems to involve the SYTANLAAF motif, especially the A7 residue.