The binding of radioactive gamma-aminobutyric acid to particulate fractions of mammalian brain was measured by a centrifugation assay, using repeatedly washed and frozen and thawed membranes and sodium-free assay conditions. Studies of over 30 structural analogues indicated an excellent correlation between inhibition of gamma-aminobutyric acid binding and activity as agonists or antagonists on gamma-aminobutyric acid synapses as reported in the neurophysiology literature. In particular, binding was inhibited by low concentrations of the gamma-aminobutyric acid receptor-specific drugs muscimol, homotaurine, and isoguvacine, but very poorly by analogues such as 2,4-diaminobutyric acid and nipecotic acid which specifically inhibit gamma-aminobutyric acid transport but are inactive on synaptic receptors. Binding was inhibited by the synaptic antagonist bicuculline but not by picro-toxinin. Thus, these binding sites show the chemical specificity required of physiological receptors for the neurotransmitter gamma-aminobutyric acid.