The NMDA-subtype of ionotropic glutamate receptor (NMDAR) is comprised of both NR1 and NR2 subunits. The NR1 subunit binds the NMDAR co-agonist glycine, while glutamate binds to NR2 subunits of which there are four types (A–D). The binding sites for either glycine or glutamate are formed by two regions within each subunit (S1 and S2) (see Chen & Wyllie, 2006). It is known that glycine potency varies between recombinant NMDARs containing different NR2 subunits, for example glycine potencies of NR2A- or NR2D-containing NMDARs differ by an order of magnitude. Therefore this heterogeneity in glycine potency must be specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is ubiquitous. To investigate the molecular mechanisms underlying this, we generated a number of chimaeric NR2A/NR2D subunits with exchanges in the S1 and S2 domains. NR2 chimaeras were coexpressed with NR1 in Xenopus oocytes and agonist-evoked inward currents examined. In the presence of saturating concentrations of glutamate, glycine concentration-response curves generated from NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC₅₀ values (EC₅₀=1.02 ± 0.06 µM) similar to wildtype NR2A-containing receptors (EC₅₀=1.29 ± 0.08 µM). However, oocytes expressing receptors containing NR2A subunits including the NR2D S2 region (EC₅₀=219 ± 13 nM) or both NR2D S1 and S2 regions (EC₅₀=233 ± 10 nM) produced glycine potencies similar to those seen in wildtype NR2D-containing NMDARs (EC₅₀=129 ± 7 nM) (n=13-26 oocytes per experiment). These changes in glycine potency occurred in the absence of any obvious alteration in competitive antagonist affinity at the glycine site, as judged by Schild analysis. This suggests that the variation in glycine potency among NMDAR subtypes may be caused by allosteric interactions between the NR1 and NR2 binding domains rather than by inducing a direct structural change in the NR1-glycine binding site.