NMDA receptors (NMDARs) are glutamate-gated ion channels that are present at most excitatory mammalian synapses. The four GluN2 subunits (GluN2A-D) contribute to four diheteromeric NMDAR subtypes that have divergent physiological and pathological roles. Channel properties that are fundamental to NMDAR function vary among subtypes. We investigated the amino acid residues responsible for variations in channel properties by creating and examining NMDARs containing mutant GluN2 subunits. We found that the NMDAR subtype specificity of three crucial channel properties, Mg(2+) block, selective permeability to Ca(2+) and single-channel conductance, were all controlled primarily by the residue at a single GluN2 site in the M3 transmembrane region. Mutant cycle analysis guided by molecular modeling revealed that a GluN2-GluN1 subunit interaction mediates the site's effects. We conclude that a single GluN2 subunit residue couples with the pore-forming loop of the GluN1 subunit to create naturally occurring variations in NMDAR properties that are critical to synaptic plasticity and learning.