Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) to an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X-100 (Triton-treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone. In contrast, Zn2+ was invariably active as a potent inhibitor of the binding irrespective of the membrane preparations used. In untreated membranes, Ca2+ markedly accelerated the initial association rate of [3H]MK-801 binding without affecting the binding at equilibrium in a manner similar to that found with glycine, as well as with glutamate; Mg2+, however, facilitated the initial association rate with a concomitant reduction of the binding at equilibrium. Zn2+ was effective in accelerating the initial rapid phase of association, with the initial slow phase being delayed, and in markedly reducing the binding at equilibrium. Both Mg2+ and Ca2+ also facilitated dissociation of the bound [3H]MK-801 and Zn2+ slowed the dissociation in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)