Abstract: Heterogeneity of binding affinities for a variety of ligands was observed for γ-aminobutyric acid type A (GABAA) receptors in the rat CNS, at both GABA and ben-zodiazepine recognition sites. Photoaffinity labeling by [3H]flunitrazepam and [3H]muscimol to affinity column-purified receptor proteins was examined by gel electropho-resis in sodium dodecyl sulfate. Anesthetic barbiturates (pentobarbital) and steroids (alphaxalone) both differentially stimulated the incorporation of [3H]flunitrazepam more so into the 51-kDa α1 subunit than into the 53-kDa aL2 polypeptide, and incorporation of [3H]muscimol into the 55-kDa β2 subunit more so than the 58-kDaβ3 polypeptide. Binding to these polypeptides was also affected differentially by other allosteric modulators and competitive inhibitors, including the benzodiazepine “type 1” selective ligand CL218.872. Heterogeneity in affinity of this drug for the single 51-kDa α1 polypeptide strongly suggests that type I receptors, like type II, are heterogeneous. In brain sections, the extent of enhancement of [3H]muscimol binding showed significant regional variation, similar for both steroids and barbiturates, and the GABA analogues THlP and taurine inhibited muscimol binding with regional variations in affinity that were almost opposites of each other. Modulation of [3H]flunitrazepam binding by steroids, barbiturates, and THlP significantly varied with regions. Taken together, ligand binding heterogeneity exhibited by photoaffinity labeling and autoradiography demonstrate the existence of multiple pharmacological-binding subtypes resulting from the combination of multiple polypeptide gene products into several oligomeric isoreceptors. Comparison of the regional distribution of binding subtypes with that of different subunit gene products allows the following conclusions about possible subunit compositions of native pharmacological receptor subtypes present in the brain: Benzodiazepine pharmacology of the oligomeric receptor isofotms is dependent on the nature of α and subunits other than α, GABA-benzodiazepine coupling is dependent on the nature of the α subunits, GABA site pharmacology is dependent on the nature of the β sub-units, and several subunits including α and β contribute to the degree of sensitivity to steroids and barbiturates. Finally, the presence of discrete subunits may be necessary but is not sufficient to postulate a defined pharmacological property.