A cDNA encoding the gamma 3 subunit of the human GABAA receptor has been obtained by molecular cloning. Its deduced amino acid sequence shows a high level of sequence identity with the published mouse and rat sequences (96%). The ligand binding pharmacology of the benzodiazepine site formed by stably-expressed human alpha 5 beta 3 gamma 2S and alpha 5 beta 3 gamma 3 GABAA receptor subtypes have been compared for a number of ligands, Benzodiazepine site ligands were found to be either non-selective or gamma 2-selective, with the exception of CL218,872, which was found to be 10-fold selective for the alpha 5 beta 3 gamma 3-containing subtype Two benzodiazepine site ligands. Ro15-4513 and FG8205 were more efficacious at alpha 5 beta 3 gamma 3 receptors than alpha 5 beta 3 gamma 2 receptors expressed in Xenopus oocytes, CL218,872, which is a partial agonist at alpha 1 containing receptors, had no intrinsic activity at either alpha 5 beta 3 gamma 2 or alpha 5 beta 3 gamma 3, alpha 1 beta 2 gamma 2S and alpha 1 beta 2 gamma 3 human GABAA receptors were also expressed in Xenopus oocytes and their benzodiazepine pharmacology investigated. Both the EC50 and efficacy of benzodiazepine site ligands were influenced by the type of gamma subunit coexpressed with alpha 1 and beta 2.