In an attempt to examine the functional significance of the molecular diversity of the N-methyl-D-aspartate (NMDA) receptor channel, we generated mutant mice defective in the epsilon 4 subunit by gene targeting technique. The epsilon 4 subunit mutant mice grew and mated normally. No epsilon 4 subunit protein was detected in the homozygous mutant mice, and the amount of the epsilon 4 subunit protein of 155 kDa was reduced in the heterozygous mice. The expressions of the other NMDA receptor channel subunit mRNAs were not appreciably affected by the mutation. The mutant mice exhibited no obvious histological abnormalities in the various brain regions and in the formation of whisker-related neuronal patterns (barrels, barreloids and barrelettes). In an open field test, however, the epsilon 4 subunit mutant mice showed a reduced spontaneous activity. No significant difference was found between the heterozygous and mutant mice in motor activity and anxiety tests. These results suggest that the epsilon 4 subunit of the NMDA receptor channel plays a role in vivo in controlling the spontaneous behavioral activity.