N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptor, which play a central role in learning, memory, and synaptic development. NMDARs are assembled as tetramers composed of two GluN1 subunits and two GluN2 or GluN3 subunits. Although NMDARs are widely expressed throughout the central nervous system, their number, localization, and subunit composition are strictly regulated and differ in a cell- and synapse-specific manner. The brain area, developmental stage, and level of synaptic activity are some of the factors that regulate NMDARs. Molecular mechanisms that control subunit-specific NMDAR function include developmental regulation of subunit transcription/translation, differential trafficking through the secretory pathway, posttranscriptional modifications such as phosphorylation, and protein-protein interactions. The GluN2A and GluN2B subunits are highly expressed in cortex and hippocampus and confer many of the distinct properties on endogenous NMDARs. Importantly, the synaptic NMDAR subunit composition changes from predominantly GluN2B-containing to GluN2A-containing NMDARs during synaptic maturation and in response to activity and experience. Some of the molecular mechanisms underlying this GluN2 subunit switch have been recently identified. In addition, the balance between synaptic and extrasynaptic NMDARs is altered in several neuronal disorders. Here, the authors summarize the recent advances in the identification of NMDAR subunit-specific regulatory mechanisms.