N-Methyl-D-aspartate receptors (NMDARs) play a central role in various physiological and pathological processes in the central nervous system. And they are commonly composed of four subunits, two GluN1 subunits and two GluN2 or GluN3 subunits. The different subunit compositions make NMDARs a heterogeneous population with distinct electrophysiological and pharmacological properties and thus with different abilities to conduct neuronal activities. The subunit composition, assembly process, and final structure of assembled NMDARs have been studied for years but no consensus has been achieved. In this study, we investigated the role of the amino terminal domain (ATD) of GluN2A in regulating NMDAR assembly. The ATD of GluN2A was first expressed in heterogeneous cells and the homodimer formation was investigated by fluorescent resonance energy transfer and non-reducing SDSPAGE electrophoresis. Each of the three cysteine residues located in the ATD was mutated into alanine, and the homodimerization of the ATD or GluN2A, as well as the heteromeric assembly of NMDARs was assessed by non-reducing SDSPAGE electrophoresis, co-immunoprecipitation and immunocytochemistry. We found that two cysteine residues, C87 and C320, in the ATD of the GluN2A subunit were required for the formation of disulfide bonds and GluN2A ATD homodimers. Furthermore, the disruption of GluN2A ATD domain dimerization had no influence on the assembly and surface expression of NMDARs. These results suggest that the two ATD domains of GluN2A are structurally adjacent in fully-assembled NMDARs. However, unlike GluN1, the homomerization of the ATD domain of GluN2A is not required for the assembly of NMDARs, implying that GluN2A and GluN1 play unequal roles in NMDAR assembly.