Three steviol glycosides, stevioside, rebaudioside A and rebaudioside C were successfully isolated and purified from the extract of leaves of Stevia rebaudiana Bertoni by high-speed counter-current chromatography (HSCCC). Based on the principle of the partition coefficient values (k) for target compounds and the separation factor (alpha) between targets compounds, the two-phase solvent system which contains n-hexane-n-butanol-water at an optimized volume ratio of 1.5:3.5:5 (v/v/v) was selected for the HSCCC separation and the lower phase was employed as the mobile phase in the head to tail elution mode. In a single operation, 200 mg of the crude extract yielded pure stevioside (54 mg), rebaudioside A (36 mg), and rebaudioside C (13 mg) with the purities of 98.3%, 98.5% and 97.6%, respectively. The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three steviol glycosides were identified by H-1 NMR, C-13 NMR and DEPT-NMR. (C) 2009 Elsevier B.V. All rights reserved.