Extrasynaptic GABA(A) receptors (eGABARs) allow ambient GABA to tonically regulate neuronal excitability and are implicated as targets for ethanol and anesthetics. These receptors are thought to be heteropentameric proteins made up of two α subunits-either α4 or α6-two β2 or β3 subunits, and one δ subunit. The GABA analog 4,5,6,7-tetrahydroisoxazolo (5,4-c)pyridin-3(-ol) (THIP) has been proposed as a selective ligand for eGABARs. Behavioral and in vitro studies suggest that eGABARs have nanomolar affinity for THIP; however, all published studies on recombinant versions of eGABARs report micromolar affinities. Here, we examine THIP sensitivity of native eGABARs on cerebellar neurons and on reconstituted GABARs in heterologous systems. Concentration-response data for THIP, obtained from cerebellar granule cells and molecular layer interneurons in wild-type and δ subunit knockout slices, confirm that submicromolar THIP sensitivity requires δ subunits. In recombinant experiments, we find that δ subunit coexpression leads to receptors activated by nanomolar THIP concentrations (EC(50) of 30-50 nM for α4β3δ and α6β3δ), a sensitivity almost 1,000-fold higher than receptors formed by α4/6 and β3 subunits. In contrast, γ2 subunit expression significantly reduces THIP sensitivity. Even when δ subunit cDNA or cRNA was supplied in excess, high- and low-sensitivity THIP responses were often apparent, indicative of variable mixtures of low-affinity αβ and high-affinity αβδ receptors. We conclude that δ subunit incorporation into GABARs leads to a dramatic increase in THIP sensitivity, a defining feature that accounts for the unique behavioral and neurophysiological properties of THIP.