GABAA receptors that contain either the α4- or α6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including β-carbolines, bind to these diazepam-insensitive receptor subtypes. Some β-carbolines [e.g. ethyl β-carboline-3-carboxylate (β-CCE) and methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM)] display a higher affinity for α4- compared to α6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric α6/α4 subunits and co-expressed these with wild-type rat β2 and γ2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (α6N204/α4I203). Substitutions at α6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive α1 subunit (S205N) resulted in a 7–8-fold reduction in affinity for the β-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the α1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by ∼19-fold and ∼38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the α subunit in diazepam-sensitive and -insensitive receptors.