The NMDA receptor opens in response to binding of NMDA and glycine. However, it remains unclear where and how gating of the NMDA receptor pore is accomplished. We show that different point mutations between S645 and I655 (thus including the highly conserved SYTANLAAF motif) of M3c in NR2B lead to constitutively open channels. The current through these constitutively open channels are readily blocked by external Mg2+ and MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate]. Also, the open-channel blocker MK-801 can no longer be trapped in these channels when NMDA and glycine are washed off. Moreover, M3c residues at or below A651(NR2B, A7 in SYTANLAAF) react with external methanethiosulfonate (MTS) reagents approximately 500 to 1000-fold faster in the presence than in the absence of agonists NMDA and glycine. In fact, the MTS modification rate shows exactly the same NMDA concentration dependence as channel activation. In contrast, those residues external to A651 are always modified with similar kinetics whether NMDA and glycine are present or not. Interestingly, MTS modification of A651C(NR2B) holds the channel constitutively open. Mutations of A651(NR2B) into arginine, tryptophan, or phenylalanine, and similar mutations of the corresponding A652 in NR1 also lead to constitutively open channels. Double-mutant cycle analysis further shows that the effects of A652(NR1) and A651(NR2B) mutations are evidently non-additive (i.e., cooperative) if mutated into residues with large side chains or with compensatory charges [e.g., A652E(NR1)+A651R(NR2B)]. The side chain of A7 thus plays a determinant role in the intersubunit distance at this level, which is directly responsible for the activation gate and activation-deactivation gating of the NMDA receptor.