NMDARs are tetrameric assemblies containing two GluN1 and two GluN2 subunits where the identity of the latter determine many of the key biophysical, pharmacological and downstream signalling properties of NMDARs. In the forebrain GluN2A and GluN2B NMDARs predominate and therefore identifying how these NMDAR subtypes contribute to various physiological and pathophysiological roles is key for our understanding of glutamatergic function. Antagonists that are sufficiently selective to block preferentially GluN2A- over GluN2B NMDARs are few in number. Recently a high throughput screen of a chemical library (Bettini et al. 2010) identified a novel antagonist, TCN 201, that showed selectivity for GluN2A over GluN2B NMDARs. We have characterized the nature of the antagonism produced by TCN 201 and used it to monitor the switch in NMDAR subunit composition in developing cortical neurones. Two-electrode voltage-clamp recordings were made from Xenopus laevis oocytes expressing recombinant GluN2A or GluN2B NMDARs. The block of GluN2A NMDARs by TCN 201 was dependent on the concentration of glycine. At a glycine concentration of 10 µM, TCN 201 blocked GluN2A currents by 82 ± 1% (n = 12) whereas a lower block of 51 ± 1 % (n = 8) was observed at a higher glycine concentration of 30 µM. Conversely, the blockade by TCN 201 was independent of the glutamate concentration used to evoke GluN2A NMDAR-mediated currents. Importantly TCN 201 (10 µM) gave minimal block of GluN2B currents (3 ± 1%, when the glycine concentration was either 10 µM or 30 µM). Schild analysis showed that TCN 201 was a potent antagonist of GluN2A NMDARs with a KB value of 70 nM; however the nature of this antagonism was not competitive as assessed by a non-linear Schild plot. We assessed the ability of TCN 201 to block NMDAR-mediated currents in rat cultured cortical neurones. At days in vitro (DIV) 7-10 NMDAR currents were strongly blocked by ifenprodil (80 ± 3%, n = 7), indicating that most NMDARs contained only GluN1 and GluN2B NMDAR subunits. At this stage TCN 201 gave only modest block of NMDAR currents (5 ± 2%). The sensitivity of the currents to ifenprodil decreased in older (DIV 14-18) cultures (57 ± 5%, n = 9) or in younger (DIV 7-10) neurones over-expressing the GluN2A subunit (24 ± 3%, n = 6), while TCN 201 gave stronger block (47 ± 4% and 16 ± 3% respectively). A negative correlation between ifenprodil and TCN 201 block was observed (R 2 = 0.91). Our data demonstrate that TCN 201 allows the monitoring of the developmental switch in NMDAR subunit composition. Future studies will use this and a related antagonist, TCN 213 (McKay et al. 2011) combined with complementary genetic approaches to investigate the physiological and pathophysiological roles played by NMDAR subtypes.