In a screening of a small library of extracts from plants of the Amazonian and Cerrado biomes, a hexane extract of Connarus tuberosus roots was found to significantly potentiate the GABA induced fluorescence in a fluorescence (FLIPR) assay in CHO cells stably expressing the α₁β₂γ₂ subtype of human GABA_A receptors. With the aid of HPLC-based activity profiling the activity was linked to the neolignan connarin. In CHO cells the activity of connarin was not abolished by increasing concentrations of flumazenil, while the effect of diazepam was increased by increasing concentrations of connarin. The effect of connarin was abolished by pregnenolone sulfate (PREGS) in a concentration-dependent manner, and the effect of allopregnanolone was further increased by increasing concentrations of connarin. In a two-microelectrode voltage clamp assay with Xenopus laevis oocytes transiently expressing GABA_A receptors composed of human α₁β₂γ₂S and α1β2 subunits connarin potentiated the GABA-induced currents, with EC₅₀ values of 1.2 ± 0.3 μM (α₁β₂γ₂S) and 1.3 ± 0.4 μM (α₁β₂), and with a maximum enhancement of currents E_max of 1959 ± 70% (α₁β₂γ₂S) and 185 ± 48% (α₁β₂). The activation induced by connarin was abolished by increasing concentrations of PREGS.