Several structural subclasses of ligands bind to the benzodiazepine (BZD) binding site of the GABA(A) receptor. Previous studies from this laboratory have suggested that imidazobenzodiazepines (i-BZDs, e.g., Ro 15-1788) require domains in the BZD binding site for high-affinity binding that are distinct from the requirements of classic BZDs (e.g., flunitrazepam). Here, we used systematic mutagenesis and the substituted cysteine accessibility method to map the recognition domain of i-BZDs near two residues implicated in BZD binding, gamma(2)A79 and gamma(2)T81. Both classic BZDs and i-BZDs protect cysteines substituted at gamma(2)A79 and gamma(2)T81 from covalent modification, suggesting that these ligands may occupy common volumetric spaces during binding. However, the binding of i-BZDs is more sensitive to mutations at gamma(2)A79 than classic BZDs or BZDs that lack a 3'-imidazo substituent (e.g., midazolam). The effect that gamma(2)A79 mutagenesis has on the binding affinities of a series of structurally rigid i-BZDs is related to the volume of the 3'-imidazo substituents. Furthermore, larger amino acid side chains introduced at gamma(2)A79 cause correspondingly larger decreases in the binding affinities of i-BZDs with bulky 3' substituents. These data are consistent with a model in which gamma(2)A79 lines a subsite within the BZD binding pocket that accommodates the 3' substituent of i-BZDs. In agreement with our experimental data, computer-assisted docking of Ro 15-4513 into a molecular model of the BZD binding site positions the 3'-imidazo substituent of Ro 15-4513 near gamma(2)A79.