Clobazam (CLB), a 1,5-benzodiazepine (BZD), was FDA-approved in October 2011 for the adjunctive treatment of seizures associated with Lennox-Gastaut syndrome (LGS) in patients 2 years and older. BZDs exert various CNS effects through allosteric modulation of GABA(A) receptors. The structurally distinct, 1,4-BZD clonazepam (CLN) is also approved to treat LGS. The precise mechanisms of action and clinical efficacy of both are unknown. Data show that the GABA(A) α(1)-subunit–selective compound zolpidem [ZOL] exhibits hypnotic/sedative effects. Conversely, data from knock-in mice carrying BZD binding site mutations suggest that the α(2) subunit mediates anticonvulsant effects, without sedative actions. Hence, the specific pattern of interactions across the GABA(A) receptor complexes of BZDs might be reflected in their clinical efficacies and adverse effect profiles. In this study, GABA(A)-receptor binding affinities of CLB, N-desmethylclobazam (N-CLB, the major metabolite of CLB), CLN, and ZOL were characterized with native receptors from rat-brain homogenates and on cloned receptors from HEK293 cells transfected with combinations of α (α(1), α(2), α(3), or α(5)), β(2), and γ(2) subtypes. Our results demonstrate that CLB and N-CLB have significantly greater binding affinities for α(2)- vs. α(1)-receptor complexes, a difference not observed for CLN, for which no distinction between α(2) and α(1) receptors was observed. Our experiments with ZOL confirmed the high preference for α(1) receptors. These results provide potential clues to a new understanding of the pharmacologic modes of action of CLB and N-CLB.