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cDNAs ENCODING NMDA receptor subunits have recently been cloned and been shown to have different distributions in theCNS. However, no studies on the possible in vivo combinations or subunit stoichiometry have yet been carried out. By combining human NR1 with rat NR2A and NR2C we have studied the pharmacological properties of three possible NMDA receptor subtypes; NR1 + NR2A, NR1 + NR2C and NR1 + NR2A + NR2C. By performing glycine concentration-response curves and comparing EC5Cs, it was possible to show that the NR1 + NR2A + NR2C receptor preferentially co-assembled when all three subunit cDNAs were present. This receptor had an affinity for glycine intermediate between that of NR1 + NR2A and NR1 + NR2C, but a similar Hill coefficient. Thus, two different NR2 subunits can combine in the same receptor, conferring unique pharmacological properties, suggesting that it is likely that two or more NR2 subunits co-assemble together in the same NMDA receptor complex.