This study investigated the binding of [3H]CGP 39653, a novel high-affinity antagonist of the N-methyl-D-aspartate (NMDA) recognition site of the NMDA receptor complex. [3H]CGP 39653 bound to the NMDA receptor in well washed rat brain membranes with an affinity of about 15 nM. Other NMDA site drugs inhibited [3H]CGP 39653 binding with the following order of potency: DL-(tetrazol-5-yl)glycine > glutamate > CGS 19755 > DL-2-amino-5-phosphonovalerate (DL-AP5) > NMDA. Glycine and 5,7-dichlorokynurenate partially inhibited binding. The polyamines spermine and spermidine increased [3H]CGP 39653 binding (EC50 values of 10 and 22 microM, respectively). This effect was mimicked by arcaine, 1,5-diethylaminopiperidine, diaminodecane, diethylenetriamine, and Mg2+. The increase in [3H]CGP 39653 was a result of an increased affinity of the binding site for the ligand with very little effect on binding site density. Spermine and Mg2+ also increased the affinity of the antagonists DL-AP5 and CGS 19755, but had only minor effects on the affinity of glutamate and NMDA. Arcaine did not reverse the enhancement of [3H]CGP 39653 binding by spermine, spermidine, or Mg2+. Channel-blocking dissociative anesthetics, including dizocilpine and ketamine, did not alter basal or Mg(2+)-stimulated [3H]CGP 39653 binding. Spermine did not alter either the enhancement of [3H]dizocilpine by glutamate or the inhibition of [3H]dizocilpine by DL-AP5 or CGS 19755. These studies show that polyamines and divalent cations selectively enhance the affinity of antagonists for the agonist binding site on the NMDA receptor complex. However, this effect is mediated by a site independent of the primary polyamine site defined using [3H]dizocilpine binding.