A compound (L-655,708) has been identified which has at least 50-fold selectivity for the benzodiazepine site on GABA_A receptors containing an α5 subunit over those containing an α1, α2, α3 or α6 subunit in combination with β3 and γ2. The compound was radiolabelled with tritium and investigated as a novel radioligand which recognizes the benzodiazepine site of GABA_A receptors which contain the α5 subunit. [³H]L-655,708 labels one saturable and specific population of binding sites in rat hippocampus with a K_d of 2.4 ± 0.7 nM and a B_max of 256 ± 42 fmol/mg protein. The pharmacology of the binding site labelled was consistent with that of receptors present in cells transfected with α5, β2 and γ2 and with receptors immunoprecipitated from rat brain with an α5-selective antiserum. It is concluded that [³H]L-655,708 is the first radioligand to date which is selective for any BZ2 subtype of the GABA_A receptor and should provide a valuable tool for elucidating the structure and function of the α5-containing GABA_A receptor subtype. Copyright © 1996 Elsevier Science Ltd