In vitro studies have suggested that the NMDA receptor consists of an essential subunit, NRl, and various modulatory NR2 subunits. To test this hypothesis directly in vivo, we generated mice carrying a disrupted NRl allele. NMDA-inducible increases in intracellular calcium and membrane currents were abolished in neurons from homozygous null mutants (NRV). Thus, NRl 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 NRZB but not NRZA occurred in NRl-/-mice, demonstrating that there is an interdependence of subunit expression. NRP 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 NRV mice appeared normal. Pathological evidence suggested that respiratory failure was the ultimate cause of death.