The NMDA receptor 3B (NR3B) subunit is the most recently identified member of the NMDA receptor family. In heterologous cells, it has been shown to reduce the Ca2+ permeability of glutamatergic receptor complexes formed together with NR1 and NR2 subunits and to form the unique excitatory glycine receptor complex with the NR1 subunit. However, it is unclear whether NR3B protein is expressed in and exerts similar functions in neurons. In addition, it is not understood how NR3B interacts with NR1 and NR2 and how such an interaction may regulate the membrane trafficking of the NMDA receptor complex. Here we report that our analysis using an antibody specific for NR3B showed that the NR3B protein is selectively expressed in somatic motor neurons in the brainstem of adult mice. Coimmunoprecipitation and electrophysiological analyses demonstrated that NR3B, when exogenously introduced into hippocampal neurons, can coassemble with endogenous NR1 and NR2A and can reduce the Ca2+ permeability of NMDA currents. In contrast, NR3B was not involved in the excitatory glycine response in neurons under our test conditions. Although NR1 or NR3B alone cannot be transported to the cell surface, coexpression of these subunits mutually supported transport of the NMDA receptor complex by interaction involving the specific regions of the C terminus of NR3B. These results indicate that NR3B may modulate the function of NMDA receptors in somatic motor neurons during adulthood by controlling membrane trafficking and by reducing Ca2+ permeability.