Two forms of GABAergic inhibition exist in the brain: a persistent tonic and transient synaptic inhibition. Mammalian GABAA receptors are hetero-pentameric, ligand-gated, chloride channels assembled from a family of & ge;19 related subunits. To test the hypothesis that tonic current in CA1 hippocampal pyramidal neurons is mediated by alpha5 subunit-containing GABA A receptors, we used an assortment of subunit-selective drugs. Previously, we showed that classical benzodiazepines enhance the tonic current. Here, we show that tonic conductance is modulated by the alpha5-selective negative modulator L-655,708 but not by the alpha5-sparing imidazopyridine, zolpidem. Moreover, beta2/3-selective positive allosteric modulators enhance the tonic current where delta-sensitive neurosteroids do not, suggesting that the alpha5 subunit assembles with beta2/3 and gamma subunits to form tonic GABA A receptor complexes in the hippocampus. Deletion of the alpha5 subunit results in a 75% reduction in tonic current. Thus, we attribute tonic inhibition in hippocampal pyramidal neurons to alpha5-containing GABA A receptors.