Activation of the N-methyl-D-aspartate (NMDA) receptor by HIV-1 envelope glycoprotein 120 (gp120) is thought to represent at least one of the pathways causing neuronal damage in AIDS patients. In the present study, recombinant gp120 binding to NMDA receptor subunits expressed in a baculovirus system was examined by immunocytochemistry and a binding assay, using horseradish peroxidase (HRP)-conjugated and 125I-labeled recombinant gp120, respectively. We found that recombinant gp120 binds to Sf21 cells expressing epsilon1/zeta1 or epsilon2/zeta1 combined NMDA receptor subunits, but not to Sf21 cells infected with mock virus or Sf21 cells expressing a single epsilon1, epsilon2, or zeta1 NMDA receptor subunit. The binding was strongly blocked by unlabeled recombinant gp120, monoclonal anti-HIV-1 gp160 antibody, and a mixture of anti-epsilon1/epsilon2 and anti-zeta1 antibodies. The same results were obtained by flow cytometric analysis. These data suggest that HIV-1 gp120 may directly bind to the NMDA receptor. This evidence enhances our understanding of the mechanism of HIV-1-induced neuronal damage in AIDS patients.