Glutamate is the major excitatory neurotransmitter of the central nervous system. NMDA-type glutamate receptors have been implicated in multiple physiological processes, including neuronal development, plasticity, and long-term potentiation. However, as seen in both acute and chronic neurodegenerative disorders, overstimulation of NMDA receptors can cause excessive Ca2+ influx, free radical generation, and abnormal enzymatic activity, and can thus contribute to synaptic injury and neuronal death. Here, we present a mechanism whereby excessive NMDA receptor activity can be preferentially abated during hypoxia and other CNS insults, but less so under physiological conditions. Under ambient air conditions, NO inhibits NMDA receptor activity via S-nitrosylation predominantly by reacting with a critical thiol group on its NR2A subunit at Cys 399 along with two additional cysteine pairs if their disulfide bonds are reduced to free thiol (these additional cysteines are on the NR1 subunit at positions 744 and 798, and on NR2 subunits at cysteine residues 87 and 320). We demonstrate that relative hypoxia enhances this S-nitrosylation reaction of NMDA receptors by a unique mechanism involving an ‘‘NO-reactive oxygen sensor motif” whose determinants include C744 and C798 of the NR1 subunit. Redox reactions involving these two thiol groups sensitize other NMDA receptor sites to S-nitrosylation and consequent receptor inhibition, while their own nitrosylation has little effect on NMDA receptor activity. The crystal structure of the ligand-binding domain of NR1 reveals a flexible disulfide bond (C744-C798), which may account for its susceptibility to reduction and subsequent reaction with NO that is observed with biochemical techniques. These thiols are S-nitrosylated preferentially during increasing hypoxia or other CNS insults, thus preventing excessive activity associated with cytotoxicity while avoiding blockade of physiologically active NMDA receptors. Takahashi H, Shin Y, Cho S-J, Zago WM, Nakamura T, Gu Z, Ma Y, Furukawa H, Liddington RL, Zhang D, Tong G, Chen H-SV, Lipton SA. Hypoxia enhances S-nitrosylation—mediated NMDA receptor inhibition via a thiol oxygen sensor motif. Neuron 2007;53:53-64.