Both native and recombinant "diazepam-insensitive" GABAA receptors (DI) are characterized by the very low affinities of prototypic 1,4-benzodiazepines such as diazepam and the high affinity of an imidazobenzodiazepine, Ro 15-4513. The presence of either an alpha 4 or alpha 6 subunit imparts this unusual pharmacological profile to DI. Based on the affinities of these compounds at recombinant DI, the pharmacological properties of alpha 4- and alpha 6-bearing receptor isoforms appear to be very similar if not identical. Using a larger sample of structurally diverse compounds, we now demonstrate distinct but related ligand binding profiles of recombinant alpha 4 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 DI. Comparison of 18 ligands drawn from three principal structural groups (beta-carbolines, imidazobenzodiazepines and pyrazoloquinolinones) revealed that the affinity of at least one representative from each group differed by > 5-fold between alpha 4- and alpha 6 beta 2 gamma 2 receptors. While the high correlation (r2 = 0.926; p < 0.001) obtained between the affinities of these ligands at alpha 4- and alpha 6-containing receptors underscores the similarity between these receptor isoforms, a significant deviation of the slope of this correlation (0.792; 95% C.I. 0.673-0.911) from unity is substantive evidence that these DI possess distinct pharmacological profiles. These findings indicate that it is feasible to develop selective ligands for these DI isoforms.