Potentiation of GABA_A receptor activation through allosteric benzodiazepine (BZ) sites produces the anxiolytic, anticonvulsant and sedative/hypnotic effects of BZs. Using a mouse line lacking α1 subunit expression, we investigated the contribution of the α1 subunit to GABA_A receptor pharmacology, function and related behaviors in response to BZ site agonists. Competitive [³H]flunitrazepam binding experiments using the Type I BZ site agonist, zolpidem, and the Type I and II BZ site non-specific agonist, diazepam, demonstrated the complete loss of Type I BZ binding sites in α1^−/− mice and a compensatory increase in Type II BZ binding sites (41±6%, P<0.002). Chloride uptake analysis in α1^−/− mice revealed an increase (108±10%, P<0.001) in the efficacy (E_max) of flunitrazepam while the EC₅₀ of zolpidem was increased 495±26% (α1^+/+: 184±56 nM; α1^−/−: 1096±279 nM, P<0.01). An anxiolytic effect of diazepam was detected in both α1^+/+ and α1^−/− mice as measured on the elevated plus maze; however, α1^−/− mice exhibited a greater percentage of open arm entries and percentage of open arm time following 0.6 mg/kg diazepam. Furthermore, α1^−/− mice were more sensitive to the motor impairing/sedative effects of diazepam (1–10 mg/kg) as measured by locomotor activity in the open field. Knockout mice were insensitive to the anticonvulsant effect of diazepam (1–15 mg/kg, P<0.001). The hypnotic effect of zolpidem (60 mg/kg) was reduced by 66% (P<0.001) in α1^−/− mice as measured by loss of righting reflex while the effect of diazepam (33 mg/kg) was increased 57% in α1^−/− mice (P<0.05). These studies demonstrate that compensatory adaptations in GABA_A receptor subunit expression result in subunit substitution and assembly of functional receptors. Such adaptations reveal important relationships between subunit expression, receptor function and behavioral responses.