Several flavonoids including 2',3,4,4'-tetrahydroxychalcone, 5,6,7,4'-tetrahydroxyflavone and butin, were separated from the seeds of Vernonia anthelmintica Willd by high-speed counter-current chromatography using a two-step operation. Two different types of solvent systems were used: chloroform-dichloromethane-methanol-water (2:2:3:2, v/v) and 1,2 dichloroethane-methanol-acetonitrile-water (4:1.1:0.25:2, v/v). From 1 kg of seeds of Vernonia anthelmintica Willd the method yielded about 45 mg of 2',3,4,4'-tetrahydroxychalcone, 40mg of 5,6,7,4'-tetrahydroxyflavone, and 55 mg of butin. Each isolated component showed 95-97% purity as determined by high-performance liquid chromatography analysis. These purified compounds were characterized by MS and NMR.