Zinc noncompetitively antagonizes N-methyl-D-aspartate (NMDA) receptor-mediated responses in cultured neurons. We investigated the mechanism of this inhibition by examining the effect of zinc on ligand binding to three distinct sites on the NMDA receptor in rat hippocampal membranes. Zinc dose-dependently inhibited both the association and dissociation of the NMDA channel blocker [3H]N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) but had no effect on steady state levels of [3H]TCP binding. This suggests that zinc inhibits the receptor-gated access of [3H]TCP to its site in the ion channel but has no effect on the binding site itself. Zinc inhibition of [3H]TCP association was not mediated by an action at the NMDA recognition site, because zinc had no effect on NMDA-displaceable L-[3H]glutamate binding. On the other hand, zinc dose-dependently inhibited [3H]glycine binding by a noncompetitive interaction. Stoichiometric analysis of equilibrium binding data indicated the presence of two [3H]glycine binding sites/[3H]TCP binding site. Comparison of the potencies of zinc in inhibiting glycine-dependent [3H]TCP association and [3H]glycine binding suggests that blockade of only one of the two glycine sites is sufficient to prevent [3H]TCP association. We hypothesize that synaptically released zinc inhibits NMDA receptor-mediated responses by binding to a site on the receptor/channel complex, reducing glycine binding, and thereby decreasing what would otherwise be a tonically present action of endogenous extracellular glycine.