The study isolated cDNA clones for four different N-methyl-D-aspartate (NMDA) receptor subunits (NMDAR2A–NMDAR2D) through PCR and molecular screening of a rat brain cDNA library. These subunits are only 15% identical to the key NMDA receptor subunit (NMDAR1) but are highly homologous (~50%) among themselves. They share large hydrophilic domains at both amino- and carboxyl-terminal sides of the four putative transmembrane segments. NMDAR2A and NMDAR2C, when expressed individually in Xenopus oocytes, showed no electrophysiological response to agonists. However, when co-expressed with NMDAR1, they significantly potentiated NMDAR1 activity and produced functional variability in agonist affinity, antagonist effectiveness, and sensitivity to Mg2+ blockade. Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many others. This investigation demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor.The study isolated cDNA clones for four different N-methyl-D-aspartate (NMDA) receptor subunits (NMDAR2A–NMDAR2D) through PCR and molecular screening of a rat brain cDNA library. These subunits are only 15% identical to the key NMDA receptor subunit (NMDAR1) but are highly homologous (~50%) among themselves. They share large hydrophilic domains at both amino- and carboxyl-terminal sides of the four putative transmembrane segments. NMDAR2A and NMDAR2C, when expressed individually in Xenopus oocytes, showed no electrophysiological response to agonists. However, when co-expressed with NMDAR1, they significantly potentiated NMDAR1 activity and produced functional variability in agonist affinity, antagonist effectiveness, and sensitivity to Mg2+ blockade. Northern blotting and in situ hybridization analyses revealed that the expressions of individual mRNAs for the NMDAR2 subunits overlap in some brain regions but are also specialized in many others. This investigation demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor.