The N-methyl-D-aspartate (NMDA) receptor channel is highly permeable to Ca2+ but is blocked by Mg2+ in a voltage-dependent manner. These characteristics are essential for the NMDA receptor channel to mediate the induction of long-term potentiation of synaptic efficacy, a form of activity-dependent synaptic plasticity thought to underlie memory, learning and development. Recent studies have revealed the molecular and functional diversity of the NMDA receptor channel subunits, which are classified into the epsilon and zeta families according to the amino-acid sequence homology. Here we report that replacement by glutamine of asparagine 598 in putative transmembrane segment M2 of the zeta 1 subunit, strongly reduces the sensitivity of the heteromeric epsilon 2/zeta 1 NMDA receptor channel to Mg2+ block. The corresponding mutation of the epsilon 2 subunit has a similar effect. Furthermore, the heteromeric epsilon 2/zeta 1 NMDA receptor channel with the mutation on both subunits shows greatly reduced sensitivity to MK-801, a channel blocker of the NMDA receptor channel, but is still susceptible to inhibition by Zn2+. These findings suggest that the conserved asparagine residue in segment M2 constitutes a Mg(2+)-block site of the NMDA receptor channel, and that the MK-801 site overlaps the Mg2+ site.