This study reports the molecular characterization of four NMDA receptor subunits (NMDAR2A–NMDAR2D) isolated from a rat brain cDNA library using PCR and molecular screening. These subunits are only about 15% identical to the key subunit of the NMDA receptor (NMDAR1) but are highly homologous (approximately 50%) with each other. They share large hydrophilic domains at both the amino- and carboxyl-terminal sides of the four putative transmembrane segments. When expressed individually in Xenopus oocytes, these subunits 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. This indicates that NMDAR1 is essential for the function of the NMDA receptor, and the multiple NMDAR2 subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDAR1. 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 other regions. The study demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor. The NMDAR2 subunits share structural similarities with other ligand-gated ion channels and possess four putative transmembrane segments. They have a peculiar structural feature of large extensions of the putative extracellular domains at both amino- and carboxyl-terminal regions, which may play a role in the diverse function and regulation of the NMDA receptor. The NMDAR2 subunits alone show no ability to respond to glutamate or NMDA, but when co-expressed with NMDAR1, they markedly potentiate NMDAR1 activity and confer functional variability in electrophysiological and pharmacological properties. The study also shows that the NMDAR2 subunits have different expression patterns in various brain regions, contributing to the functional diversity of the NMDA receptor. The findings highlight the importance of NMDAR1 as a key subunit for the NMDA receptor-channel complex and the role of NMDAR2 subunits in generating functional diversity through heteromeric configurations with NMDAR1. The study provides insights into the complex mechanisms of glutamate neurotransmission, synaptic plasticity, and neurotoxicity, as well as the molecular mechanisms of ion permeation and functional modulation characteristic of the NMDA receptor.This study reports the molecular characterization of four NMDA receptor subunits (NMDAR2A–NMDAR2D) isolated from a rat brain cDNA library using PCR and molecular screening. These subunits are only about 15% identical to the key subunit of the NMDA receptor (NMDAR1) but are highly homologous (approximately 50%) with each other. They share large hydrophilic domains at both the amino- and carboxyl-terminal sides of the four putative transmembrane segments. When expressed individually in Xenopus oocytes, these subunits 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. This indicates that NMDAR1 is essential for the function of the NMDA receptor, and the multiple NMDAR2 subunits potentiate and differentiate the function of the NMDA receptor by forming different heteromeric configurations with NMDAR1. 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 other regions. The study demonstrates the anatomical and functional differences of the NMDAR2 subunits, which provide the molecular basis for the functional diversity of the NMDA receptor. The NMDAR2 subunits share structural similarities with other ligand-gated ion channels and possess four putative transmembrane segments. They have a peculiar structural feature of large extensions of the putative extracellular domains at both amino- and carboxyl-terminal regions, which may play a role in the diverse function and regulation of the NMDA receptor. The NMDAR2 subunits alone show no ability to respond to glutamate or NMDA, but when co-expressed with NMDAR1, they markedly potentiate NMDAR1 activity and confer functional variability in electrophysiological and pharmacological properties. The study also shows that the NMDAR2 subunits have different expression patterns in various brain regions, contributing to the functional diversity of the NMDA receptor. The findings highlight the importance of NMDAR1 as a key subunit for the NMDA receptor-channel complex and the role of NMDAR2 subunits in generating functional diversity through heteromeric configurations with NMDAR1. The study provides insights into the complex mechanisms of glutamate neurotransmission, synaptic plasticity, and neurotoxicity, as well as the molecular mechanisms of ion permeation and functional modulation characteristic of the NMDA receptor.