The modulation of solubilized type 1 and type 2 benzodiazepine receptors from cow brain by gamma-aminobutyric acid (GABA), divalent cations, and anions has been evaluated. GABA stimulates [3H] flunitrazepam binding of both receptor subtypes, whereas divalent cations and anions selectively stimulate solubilized type 2 receptors. Of numerous anions examined, only chloride, bromide, and iodide enhance [3H] flunitrazepam binding. Chloride and bromide increase mainly receptor affinity for [3H]flunitrazepam, whereas iodide largely influences Bmax values. Divalent cations also stimulate soluble type 2 receptors. Calcium, zinc, manganese, barium, and magnesium have similar potencies in enhancing [3H]flunitrazepam binding, whereas copper and nickel are about 4 to 5 times more potent. The 2- to 3-fold increase in type 2 receptor binding by divalent cations involves change in numbers of binding sites. Effects of combinations of GABA, calcium, and chloride suggest that they may exert their modulating effects on type 2 receptors through different mechanisms. GABA, calcium, and chloride also protect [3H]flunitrazepam binding from heat inactivation, indicating a close link in the native state between the GABA, ions, and the benzodiazepine recognition sites. Since physiologic concentrations of calcium and chloride influence type 2 receptors, these ions may be involved in those pharmacologic effects of benzodiazepines mediated by type 2 sites.