Using a baculovirus expression vector system, the ζ1 subunit of the mouse N-methyl-d-aspartate (NMDA) receptor channel was expressed in Spodoptera frugiperda insect cells. The peptide corresponding to the C-terminus of the ζ1 subunit was synthesized by using the multiple antigen peptide (MAP) system, and an antibody to the synthetic peptide was produced. Immunoblotting using the newly developed antibody revealed the major 122-kDa and the minor 104-kDa protein bands. The effect of tunicamycin on the immunoblots and [ 3 5 S]methionine/[ 3 5 S]cysteine metabolic radiolabeling suggested that the two bands corresponded to glycosylated and non-N-glycosylated forms, respectively. Membranes prepared from insect cells infected with the recombinant virus had the binding activity of antagonist ligand 5,7-[3- 3 H]dichlorokynurenate (DCKA) of a glycine recognition domain of the receptor. Both immunofluorescence labeling and the [ 3 H]DCKA binding assays also showed a greater level of expression (B m a x = 51 pmol/mg protein) in the insect cells. The ligand binding characteristics of the receptors expressed in insect cells suggested that the single ζ1 subunit protein has glycine antagonist binding properties comparable to those of the native NMDA receptor channels. The lack of DCKA-binding activity of the non-N-glycosylated NMDA receptor expressed in the presence of tunicamycin suggested that N-linked oligosaccharide is essentially required for expression of a functional receptor in insect cells. This is the first report describing the importance of N-glycosylation for the acquisition of ligand binding to NMDA receptor channel subunit protein.