The higher order structure of antibacterial polymyxin B (PxB), an N-acylated pentacationic (4-10)-cyclic decapeptide, is determined from NMR data by simulated annealing calculations. The antibacterial selectivity of PxB against Gram-negative organisms suggests that PxB must participate in specific microscopic interactions with these organisms, and the structure of PxB provides insights into these interactions. Significance of the topological flexibility of certain parts of the structure in relation to the membrane-mimetic environment is developed to suggest the presence of two distinct and specific phosphoester binding sites per PxB. Although disordered in water, PxB remains in a monomeric form and adopts a well-defined structure in aqueous trifluoroethanol (TFE). Circular dichroism results show a comparable structure in aqueous TFE and on anionic vesicles. Docking and energy minimization calculations show that the two phosphoester binding sites are essentially on the same face of the structure. The topology of the ring is locked in a fixed relationship between residues 6, 7, and 10. However, the pucker of the ring changes residues 4 and 5 on one side and residues 8 and 9 on the other side. The structural flexibility within the NMR constraints permits occupancy of the sites individually or simultaneously, in a 10 to 14 Angstrom range for the phosphorus-to-phosphorus distance between the two sites. Thus, for interactions at the Gram-negative cell surface, a PxB molecule could not only bind to the headgroup of one or two phosphatidylglycerols, but remarkably, the two sites could also simultaneously accommodate the 1,4'-diphosphodiglucosamine of lipid A backbone of a lipopolysaccharide. The observed combination of both fixed and flexible regions of PxB, referred to as higher order structure, accounts for its ability to perform a range of microscopically distinct functions guided by the local environment at the bacterial cell surface.