The interaction of Ro 25–6981 with N-methyl-d-aspartate (NMDA) receptors was characterized by a variety of different testsin vitro. Ro 25–6981 inhibited ³H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC₅₀ values of 0.003 μM and 149 μM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC₅₀ values of 0.009 μM and 52 μM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25–6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25–6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 μM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC₅₀ values of 0.4 μM and 0.04 μM, respectively. Ro 25–6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 μM for 20 h) and only weak activity in blocking Na⁺ and Ca⁺⁺ channels, activated by exposure of cortical neurons to veratridine (10 μM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25–6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.