A method of using high-speed counter-current chromatography (HSCCC) was established for preparative isolation and purification of antimycin A components from antimycin fermentation broth. Six antimycin A components were successfully purified for the first time by HSCCC with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (5:2:4:1, by volume). Total of 20mg antimycin A(4)(a or b), 25mg antimycin A(3)(a or b), 21mg antimycin A(8)(a or b), 34mg antimycin A(2)(a or b), 26mg antimycin A(1)(a or b) and 34mg antimycin A(1)(a or b) with the purities of 93.2, 98.6, 96.2, 94.1, 94.9 and 96.7%, respectively, determined by high-performance liquid chromatography (HPLC), were yielded from 200mg crude sample only in one HSCCC run.