NMDA receptors (NMDARs) are heteromeric assemblies of NR1 and NR2(A-D) subunits with properties heavily influenced by the type of NR2 subunit incorporated. While NMDARs with only one type of NR2 subunit have been extensively characterized, little is known about receptors containing two different NR2 subunits, despite compelling evidence that such triheteromeric receptors exist in vivo. We used a point-mutation approach that allows isolation of recombinant triheteromeric NMDARs possessing two different NR2 N-terminal domains (NTDs). We show that in receptors associating the NR2A-NTD (sensing nanomolar Zn) and the NR2B-NTD (sensing ifenprodil), each NTD binding site retains selective high affinity for its ligand. However, each ligand produces only partial inhibition, and maximal inhibition requires occupancy of both NR2-NTDs by their respective ligands. Similarly, NR1/2A/2C receptors are inhibited by zinc with high potency but low efficacy. Therefore, interactions between homologous N-terminal domains determine the unique pharmacological properties of triheteromeric NMDARs.