AbstractGABAAreceptors are the main inhibitory neurotransmitter receptors in the brain and are targets for numerous clinically important drugs such as benzodiazepines, anxiolytics and anesthetics. We previously identified novel ligands of the classical benzodiazepine binding pocket in α1β2γ2GABAAreceptors using an experiment-guided virtual screening (EGVS) method. This screen also identified novel ligands for intramembrane low affinity diazepam site(s). In the current study we have further characterized compounds 31 and 132 identified with EGVS as well as 4-O-methylhonokiol. We investigated the site of action of these compounds in α1β2γ2GABAAreceptors expressed inXenopus laevisoocytes using voltage-clamp electrophysiology combined with a benzodiazepine site antagonist and transmembrane domain mutations. All three compounds act mainly through the two β+/α−subunit transmembrane interfaces of the GABAAreceptors. We then used concatenated receptors to dissect the involvement of individual β+/α−interfaces. We further demonstrated that these compounds have anesthetic activity in a small aquatic animal model,Xenopus laevistadpoles. The newly identified compounds may serve as scaffolds for the development of novel anesthetics.