In South Africa Rhus pyroides is traditionally used in the treatment of epilepsy. In the present study two biflavonoids with activity in the (3)H-Ro 15-1788 (flumazenil) binding assay were isolated by high pressure liquid chromatography (HPLC) fractionation of the ethanol extract of the leaves from Rhus pyroides. The structures of the two biflavonoids were elucidated by nuclear magnetic resonance spectroscopy (NMR) to be agathisflavone and amentoflavone. Agathisflavone and amentoflavone competitively inhibited the binding of (3)H-Ro 15-1788 with a K(i) of 28 and 37 nM, respectively. Extracts of Rhus dentata and Rhus pentheri were not as active as the extract from Rhus pyroides; both were found to contain apigenin and agathisflavone. The monomer apigenin, agathisflavone and amentoflavone were fitted into a pharmacophore model for ligands binding to the GABA(A) receptor benzodiazepine site. This reflected the affinities of the compounds in the [(3)H]-flumazenil binding assay.