N-Methyl-D-aspartate (NMDA)â€“type glutamate receptors mediate excitatory synaptic transmission in the central nervous system and play critical roles in many neuronal processes. The physiologic roles of NMDA receptors are shaped by their functional properties, which are highly dependent on subunit composition. Most NMDA receptors are assembled from two GluN1 and two GluN2 subunits, but diversity in subunit composition is made possible by eight GluN1 splice variants (i.e., isoforms) and four distinct GluN2 subunits (GluN2A-D). We demonstrate using FÃ¶rster resonance energy transfer and fluorescence lifetime imaging that GluN1-1a and GluN1-1b isoforms, which include or lack residues encoded by exon 5, form triheteromeric GluN1-1a/GluN1-1b/GluN2A (1a/1b/2A) and GluN1-1a/GluN1-1b/GluN2B (1a/1b/2B) receptors. We describe the selective expression of NMDA receptors containing two different GluN1 isoforms, and show that triheteromeric 1a/1b/2A and 1a/1b/2B receptors exhibit intermediate deactivation kinetics and pharmacological properties compared with the respective diheteromeric GluN1-1a/GluN1-1a/GluN2 and GluN1-1b/GluN1-1b/GluN2 receptors. These results highlight the intriguing possibility that neurons can finely tune NMDA receptor signaling by shifting the ratio of expressed GluN1-1a and GluN1-1b isoforms. Furthermore, we evaluate the contribution of channel pore residues to magnesium block and calcium permeability. These data point to the asymmetric contribution of pore residues in GluN1 and GluN2 to magnesium block, and reveal that a single copy of pore residues from GluN3 subunits strongly attenuates magnesium block and calcium permeability of NMDA receptors. Thus, the selective expression of NMDA receptors containing two distinct GluN1 isoforms provides new opportunities to study functional properties relevant to neuronal receptors.