The primary structure of a novel subunit of the mouse NMDA (N-methyl-D-aspartate) receptor channel, designated epsilon4, has been revealed by cloning and sequencing the cDNA. The epsilon4 subunit shares high amino acid sequence identity with the epsilon1, epsilon2 and epsilon3 subunits of the mouse NMDA receptor channel, thus constituting the epsilon subfamily of the glutamate receptor channel. Expression from cloned cDNAs of the epsilon4 subunit together with the zeta subunit in Xenopus oocytes yields functional NMDA receptor channels. The epsilon4/zeta1 heteromeric channel exhibits high apparent affinities for agonists and low sensitivities to competitive antagonists. The epsilon4 subunit is thus distinct in functional properties from the epsilon1, epsilon2 and epsilon3 subunits, and contributes further diversity of the NMDA receptor channel.