We investigated the recognition properties of different GABA(A) receptor subtypes and mutant receptors for the biflavonoid amentoflavone, a constituent of St. John's Wort. Radioligand binding studies showed that amentoflavone recognition paralleled that of the classical benzodiazepine diazepam in that it had little or no affinity for alpha4- or alpha6-containing receptors. Lysine and alanine substitutions at position 101 of the rat alpha1 subunit resulted in a complete loss of competitive amentoflavone binding, but functional analysis of the alanine mutant expressed with beta2 and gamma2 subunits in Xenopus oocytes revealed no significant difference in the negative modulation of GABA-induced currents brought about by amentoflavone. Furthermore, elimination of the gamma subunit had no effect on the negative modulation of these currents. This negative modulation was also observed at alpha1beta1gamma2 GABA(A) receptors and is therefore not likely mediated by the loreclezole site. These results suggest a complex mechanism of amentoflavone interaction at GABA(A) receptors.