The catalytic subunit of the serine–threonine protein phosphatase 2A (PP2A) was previously found to bind to the carboxyl domain of NMDA receptor (NMDAR) subunit NR3A. We now report that NR3A constitutively associates with the PP2A holoenzyme, but not the core enzyme in rat brain synaptic plasma membranes. We also identified critical amino acids in NR3A required for binding to PP2A. We performed alanine-scanning mutagenesis in the PP2A-binding domain of the NR3A C-terminal (NR3Ac), then co-expressed the mutants together with the PP2A catalytic subunit in a yeast two-hybrid system and human embryonic kidney (HEK) 293 cells. We found that mutation of leucine 958, leucine 973 or histidine 974 or deletion of a spacer sequence of more than six amino acids between leucine 958 and histidine 974 disrupted the NR3A/PP2A interaction.