The N-methyl-D-aspartate (NMDA) receptor has been implicated in the formation of synaptic connections. To investigate the role of the epsilon2 (NR2B) NMDA receptor subunit, which is prominently expressed during early development, we used neurons from mice lacking this subunit. Although epsilon2(-/-) mice die soon after birth, we examined whether NMDA receptor targeting to the postsynaptic membrane was dependent on the epsilon2 subunit by rescuing hippocampal neurons from these mice and studying them in autaptic cultures. In voltage-clamp recordings, excitatory postsynaptic currents (EPSCs) from epsilon2(-/-) neurons expressed an NMDA receptor-mediated EPSC that was apparent as soon as synaptic activity developed. However, compared with wild-type neurons, NMDA receptor-mediated EPSC deactivation kinetics were much faster and were less sensitive to glycine, but were blocked by Mg(2+) or AP5. Whole cell currents from epsilon2(-/-) neurons were also more sensitive to block by low concentrations of Zn(2+) and much less sensitive to the epsilon2-specific antagonist ifenprodil than wild-type currents. The rapid NMDA receptor-mediated EPSC deactivation kinetics and the pharmacological profile from epsilon2(-/-) neurons are consistent with the expression of zeta1/epsilon1 diheteromeric receptors in excitatory hippocampal neurons from mice lacking the epsilon2 subunit. Thus epsilon1 can substitute for the epsilon2 subunit at synapses and epsilon2 is not required for targeting of NMDA receptors to the postsynaptic membrane.