Commercial colistin (polymyxin E)-complex was separated into two major components (colistins A and B) on a preparative scale by HPLC (alkyl bonded silica and aqueous-organic mobile phase containing 0.2 M NaCl-HCl buffer (pH 2.0)). Desalting of the colistins A and B fractions was completed by reversed-phase adsorption and elution using methyl alcohol. In these experiments, it was inadvertently found that prolonged elution with water gave two hydrophilic peptides, which were tentatively named colistins A_H and B_H, respectively. Further elution with methyl alcohol produced two lipophilic peptides which were named colistins A_L and B_L. Colistin BH showed higher potency than colistin A_H, A_L or B_L, and it was also effective in vivo. The fatty acid and amino acid composition of colistin B_H was identical with that of colistin B_L, but colistin B_A contained a relatively large amount of nonionic sodium which scarcely responded to the sodium ion-selective electrode of an ion meter; colistin B_H had a slightly lower molar extinction coefficient than the colistin B_L which contained negligible amount of nonionic sodium. Colistin A_H also contained nonionic sodium. Potassium containing colistins could also be derived from colistin-complex. These compounds could not be formed merely by adding sodium chloride or potassium chloride to colistin-complex solution. To obtain the sodium-containing compounds, contact with some hydrophobic stationary phase, such as alkyl-bonded silica or styrene-divinylbenzene copolymer, was necessary. It is postulated that one of the antibacterial mechanisms of polymyxin is associated with its complex-forming action on monovalent cations after contact with the lipid layer of the bacterial outer membrane.