N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.