The N-Methyl-D-Aspartate type of glutamate receptor (NMDAR) plays central roles in normal and pathological neuronal functioning. We have examined the regulation of the NR1 subunit of the NMDAR in response to excessive activation of this receptor in in vitro and in vivo models of excitotoxicity. NR1 protein expression in cultured cortical neurons was specifically reduced by stimulation with 100 µM NMDA or Glutamate. NMDA decreased NR1 protein amounts by 71% after 8 h. Low NMDA concentrations (=10 µM) had no effect. NR1 down-regulation was inhibited by the general NMDAR antagonist DL-AP5 and also by ifenprodil, which specifically antagonizes NMDARs containing NR2B subunits. Arrest of NMDAR signaling with DL-AP5 after brief exposure to NMDA did not prevent subsequent NR1 decrease. Down-regulation of NR1 did not involve calpain cleavage, but resulted from a decrease in de novo synthesis consequence of reduced mRNA amounts. In contrast, NMDA did not alter the expression of NR2A mRNA or newly synthesized protein. In neurons transiently transfected with an NR1 promoter/luciferase reporter construct, promoter activity was reduced by 68% after 2 h stimulation with NMDA, and its inhibition required extracellular calcium. A similar mechanism of auto-regulation of the receptor probably operates during cerebral ischemia, since NR1 mRNA and protein were strongly decreased at early stages of blood reperfusion in the infarcted brains of rats subjected to occlusion of the middle cerebral artery. Since NR1 is the obligatory subunit of NMDARs, this regulatory mechanism will be fundamental to NMDAR functioning.