We have quantified the effects of the NMDA receptor antagonist, (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077; (1)), at rat recombinant NR1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either pre-applied or co-applied with glutamate. The IC₅₀ values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses being 31 ± 2 nM (n=12; with glutamate at its EC₅₀ concentration) and 214 ± 10 nM (n=10; at 10-times the EC₅₀ concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K_B) of 15 ± 2 nM (n=9). Furthermore, Schild analysis of an NMDA receptor carrying a threonine to alanine point mutation in the NR2A ligand-binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K_B increased by around 15-fold (220 ± 10 nM; n=8). At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC₅₀ value of 215 ± 13 nM (n=14) was obtained in the presence of the EC₅₀ concentration of glutamate (1.5 μM), while a value of 2.2 ± 0.14 μM (n=8) was obtained with higher (15 μM) glutamate concentrations. Schild analysis gave a K_B for NVP-AAM077 at NR2B-containing receptors of 78 ± 3 nM. In the case of both NR1/NR2A and NR1/NR2B NMDA receptors the antagonism by NVP-AAM077 was surmountable by increasing the concentration of glutamate used to evoke responses. Thus while our data show that NVP-AAM077 is indeed a more potent antagonist at NR1/NR2A compared to NR1/NR2B NMDA receptors the extent of its selectivity is only around 5-fold. Using a kinetic scheme (2) to model ‘synaptic-like’ activation (10 mM pulse of glutamate for 1 ms) of NMDA receptors we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate selectively between NR2A-containing or NR2B-containing NMDA receptors and that substantial block of both NMDA receptor subtypes occurs with concentrations of NVP-AAM077 commonly used in neurophysiological studies. Indeed the potency of NVP-AAM077 at both NR1/NR2A and NR1/NR2B NMDA receptors is such that on a ‘synaptic time-scale’ this antagonist can be considered to act in an irreversible manner.