NMDA receptors are widely distributed throughout the central nervous system and are critical for neuronal development. The precise trafficking and localization of NMDA receptors at excitatory synapses is essential for proper neurotransmission and synaptic plasticity. Endogenous NMDA receptors are tetramers composed of two NR1 subunits, which are essential components of NMDA receptors, combined with additional NR2 subunits (NR2A-D). Whereas the NR1 subunit is expressed throughout the brain, the NR2 subunits display unique spatiotemporal distributions and they confer distinct functional and pharmacological properties on NMDA receptors. We have investigated the molecular determinants encoded within the NR2A, NR2B, and NR2C subunits that regulate trafficking and surface expression of NMDA receptors. All NR2 subunits possess very long intracellular C-termini that contain important regulatory motifs including protein phosphorylation sites and protein-protein interaction domains. We have examined the distal C-terminus of NR2A-C and characterized regulatory motifs in this region of each of these proteins. Although all three contain a PDZ binding domain and interact directly with the PSD-95 family of proteins, just upstream of the PDZ ligand, the sequences are quite divergent suggesting molecular specializations corresponding to these regions. The NR2A and NR2B subunits are highly expressed in the hippocampus and cortex. Interestingly, the expression of these subunits is developmentally regulated such that NR2B is expressed early in development, whereas NR2A expression is delayed. NR2B contains a strong consensus tyrosine-based endocytic motif just upstream of the PSD-95 binding site. We find this motif regulates a direct interaction with the medium chain of the AP-2 adaptor complex and is important for NR2B endocytosis (Lavezzari et al. 2003). Although the tyrosine is conserved in NR2A, the surrounding residues are distinct resulting in much lower affinity for the AP-2 medium chain. Furthermore, we find that NR2A and NR2B are differentially sorted following endocytosis, with NR2B-containing NMDA receptors preferentially trafficking through recycling endosomes (Lavezzari et al. 2004). These findings demonstrate that subunit-composition determines the intracellular sorting and surface expression of NMDA receptors. Unlike NR2A and NR2B, the NR2C subunit is specifically enriched in the cerebellum. The expression of NR2C is developmentally regulated and only appears in adult. Interestingly, NR2C does not have the tyrosine-based endocytic motif, but contains a strong prototypical PKA consensus motif just upstream from the PDZ binding site. We find that the serine within this consensus sequence (serine 1244) is robustly phosphorylated by PKA and PKC, both in vitro and in vivo, but that the phosphorylation does not affect binding of the PSD-95 family of proteins or surface expression of NR2C-containing receptors. However, we surprisingly find that modification of serine 1244 regulates the channel kinetics of NR1/NR2C NMDA receptors expressed in heterologous cells. In conclusion, the unique NR2 C-termini contain distinct substrates for the endocytic machinery and for protein kinases, demonstrating that this region of the NR2 subunits of NMDA receptors provides diversity in NMDA receptor regulation and trafficking. These defined regulatory motifs modulate a variety of NMDA receptor properties including receptor trafficking, surface expression, and, in the case of the NR2C subunit, channel kinetics.