The present study tested the hypothesis that nitration is a mechanism of hypoxia-induced modification of the N-methyl-D-aspartate (NMDA) receptor. To test this hypothesis the effect of hypoxia on the nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor was determined. Furthermore, the effect of administration of a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNLA) on the hypoxia-induced nitration of the NMDA receptor subunits as well as the NMDA receptor-mediated Ca²⁺ influx, an index of NMDA receptor–ion channel function, were determined in cortical synaptosomes. Studies were performed in newborn piglets divided into normoxic, hypoxic and hypoxic-NNLA groups. Hypoxia was induced by decreasing the FiO₂ to 0.07–0.09 for 60 min. Cerebral tissue hypoxia was confirmed by determining the levels of high energy phosphates ATP and phosphocreatine. Nitration of the NMDA receptor subunits was determined by immunoprecipitation using specific antibodies and western blot analysis. NMDA receptor–ion channel-mediated Ca²⁺ influx was determined using ⁴⁵Ca²⁺. There was a significant increase in the nitrated NR1, NR2A and NR2B subunits following hypoxia: 104±11 vs. 275±18 optical density (OD)×mm² for NR1 (P<0.05), 212±36 vs. 421±16 OD×mm² for NR2A (P<0.05) and 246±44 vs. 360±26 OD×mm² for NR2B (P<0.05). This increase in nitrated NR1, NR2A and NR2B subunits of the NMDA receptor was prevented by the administration of NNLA prior to hypoxia (NR1 160±19, P=NS, NNLA vs. normoxic; NR2A 304±49, P=NS, NNLA vs. normoxic, and NR2B 274±19, P=NS, NNLA vs. normoxic). The increase in nitration of the NR1, NR2A and NR2B subunits of the NMDA receptor increased as a function of decreased cerebral high-energy phosphates, ATP and phosphocreatine, during hypoxia. Furthermore, NOS blockade prior to hypoxia resulted in prevention of the hypoxia-induced increase in NMDA receptor-mediated Ca²⁺ influx. Our results demonstrate that hypoxia results in increased nitration of the NMDA receptor subunits and that administration of an NOS inhibitor prior to hypoxia prevents the hypoxia-induced nitration of the NMDA receptor subunits as well as the hypoxia-induced increase in NMDA receptor-mediated Ca²⁺ influx. We conclude that nitration is a mechanism of modification of the NMDA receptor function during hypoxia in the newborn piglet brain.