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The radiochemical synthesis and pharmacological properties are described of [3H]RY 80 (ethyl-8-acetylene-5, 6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1, 4]benzodiazepine-3-carboxylate, [ethyl-3H]). This compound is one of a series of 8-substituted imidazobenzodiazepines that exhibits both high affinity and selectivity for gamma-aminobutyric acid (GABA)A receptors containing alpha-5 subunits. Saturable, high-affinity (Kd approximately 0.7 nM) binding of [3H]RY 80 was observed in hippocampal membranes. The maximum number (Bmax) of [3H]RY 80 binding sites was approximately 18% of that obtained with [3H]flunitrazepam, a radioligand that labels all "diazepam-sensitive" GABAA receptors. This value is consistent with previous estimates (10-20%) of the proportion of rat hippocampal GABAA receptors containing alpha-5 subunits determined by immunoprecipitation with selective antibodies and competition experiments using an alpha-5-selective ligand. In recombinant GABAA receptors composed of alpha-5 beta-3 gamma-2 subunits, the Kd of [3H]RY 80 (approximately 0.5 nM) was consistent with the value obtained in hippocampus, whereas the Bmax value was not significantly different from that obtained with [3H]flunitrazepam. The potencies of several benzodiazepine site ligands to inhibit [3H]RY 80 binding to hippocampal membranes were in agreement with the values obtained in recombinant (alpha-5 beta-3 gamma-2) GABAA receptors. [3H]RY 80 was used both in a "GABA shift" assay to correctly predict the in vivo actions of a novel, alpha-5-selective ligand and to characterize a population of GABAA receptors containing alpha-5 subunits in neonatal rat cortex. These findings demonstrate that [3H]RY 80 can be used as a radioligand to examine the properties of GABAA receptors containing alpha-5 subunits.