In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NR1, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NR I allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants ( NR1 −/− ). Thus, NR1 has a unique role, which cannot be substituted by any other subunit, in determining the activity of the endogenous NMDA receptor. A concomitant reduction in levels of NR2B but not NR2A occurred in NR1 −/− mice, demonstrating that there is an interdependence of subunit expression. NR1 −/− mice died 8–15 hr after birth, indicating a vital neonatal function for the NMDA receptor. Although the NMDA receptor has been implicated in several aspects of neurodevelopment, overall neuroanatomy of NR1 −/− mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.