Binding to gamma-aminobutyric acid-A (GABAA) receptors was studied in synaptosomal membranes of rat brain. Dissociation of [3H]muscimol and the GABAA antagonist [3H]2-(3-carboxypropyl)-3-amino-6-p-methoxyphenylpyridazinium bromide ([3H]SR 95531) binding elicited by 100-fold dilution was accelerated by excess of GABA or SR 95531. Control dissociation might be retarded by rebinding. The contribution of a rapid first phase of dissociation of the agonist [3H]muscimol was preferentially enhanced by SR 95531. In contrast, the dissociation of [3H]SR 95531 binding was preferentially accelerated by GABA. These opposite preferential accelerations can be explained by negative heterotropic cooperativity and a reversed affinity relationship of agonists and antagonists to GABAA binding sites with different affinities. Modification of the membranes by p-diazobenzenesulfonic acid (DSA) selectively decreased the accelerating effect of GABA on the dissociation of [3H]SR 95531 binding. [3H]Strychnine binding was studied in a membrane preparation of rat spinal cord. The dissociation of the antagonist [3H]strychnine elicited by dilution was preferentially accelerated by glycine. Again, pretreatment with DSA decreased selectively this negative heterotropic (i.e., allosteric) interaction. Chemical modification by DSA might be attributed to tyrosine residues responsible for similar allosteric interactions for the GABA- and glycine-gated chloride channels.