In this study, we investigated the hypothesis that inhibition of the N-methyl-D-aspartate (NMDA) receptor complex by zinc involves a polyamine-sensitive regulatory site. We found that the specific binding of the open channel ligand [3H]MK-801 to rat hippocampal membranes 1) was inhibited by low concentrations of Zn2+ (IC50 = 5.5 microM) by 65%. 2) This high-affinity component of inhibition was reversed by the polyamine spermine to an extent that could be reconciled with competitive interaction between Zn2+ and spermine. 3) Partial inhibition by Zn2+ was additive with partial inhibition by ifenprodil, an inhibitor of the NMDA receptor complex supposed to act at a polyamine-sensitive regulatory site, and 4) in membranes prepared from several other brain regions, inhibition of [3H]MK-801 binding by Zn2+ and by ifenprodil was either less than additive, or superadditive. Our observation that ifenprodil, at concentrations saturating its high-affinity component of inhibition, prevented spermine from reversing the inhibition by Zn2+ indicates that spermine did not increase [3H]MK-801 binding by competition with Zn2+ but rather via another polyamine regulatory site not sensitive to zinc but sensitive to ifenprodil. We conclude that Zn2+ reduces channel opening of the NMDA receptor complex by allosteric inhibition of a polyamine-sensitive regulatory site different from that inhibited by ifenprodil and that these two allosteric sites influence each other in a manner dependent on the brain region investigated. The different proportions of zinc/ifenprodil inhibition in different regions could reflect different percentages of various NMDA receptor subtypes.