[3H]TCP and [3H]ifenprodil binding to N-methyl-D-aspartate (NMDA) receptors in rat forebrain membranes was used to compare the inhibition of haloperidol and trifluperidol with that of ifenprodil and eliprodil. In the [3H]TCP binding assay, inhibition curves of ifenprodil, eliprodil, haloperidol and trifluperidol revealed two affinity states in the presence of glutamate, glycine and spermidine. The potency of these agents to inhibit the high-affinity fraction of the binding agreed with the results of other studies investigating their potency to block glutamate-induced current at recombinant NR1a/NR2B NMDA receptors expressed in Xenopus oocytes. These agents also inhibited [3H]ifenprodil binding in a biphasic manner, whether in the absence or the presence of either the sigma site ligand GBR-12909 or spermidine. Spermidine reduced the fraction of high-affinity sites labeled with [3H]ifenprodil. The only alteration in the affinity was a decrease in the IC50 value of haloperidol to inhibit the high-affinity fraction of [3H]ifenprodil binding. GBR-12909 also reduced the fraction of [3H]ifenprodil sites inhibited by these compounds with high affinity, with no change in the affinity for either fraction. These data suggest that spermidine is neither a competitive antagonist at the fraction of the binding inhibited by these agents with high affinity, nor is this fraction of the binding to sigma sites. Haloperidol and trifluperidol represent a new class of agent that interacts at a site that is labeled by [3H]ifenprodil as well as [3H]TCP in rat brain membranes and that closely reflects ifenprodil's voltage-independent site on the recombinant NR1a/NR2B subtype of the NMDA receptor.