Two polymyxin antibiotics, polymyxins B and E (colistin), have been separated analytically into ten to thirteen components on a commercial reversed-phase material by isocratic elution with a mixture of acetonitrile, phosphate/formate and acetate buffer containing sodium sulphate and triethylamine. The analytical system was transferred to a preparative system, using a C18-bonded stationary phase, without extensive impairing of the selectivity. The major components of each product were isolated and characterized by high-performance liquid chromatography, amino acid analysis and identification of the fatty acid. Three components were isolated and characterized for the first time. The fatty acid was also identified in some of the minor components.