A high-speed counter-current chromatography (HSCCC) method was developed for preparative isolation and purification of alkaloids from Tripterygium Wilfordii Hook F. Preparative HSCCC with a two-phase solvent system composed of petroleum ether-ethyl acetate-ethanol-water (6:4:5:8, v/v/v/v). The organic phase was used as the stationary phase of HSCCC, and the aqueous phase as the mobile phase. Seven hundred milligrams total alkaloids yielded 210 mg of wilfortrine, 96 mg of wilfordine, 220 mg of wilforgine and 100 mg of wilforine, with the purity of 90.3%, 92%, 99.5% and 93.5% in one step separation, respectively. (C) 2007 Elsevier B.V. All rights reserved.