Polymyxin nonapeptides such as polymyxin B nonapeptide (PMBN) are polymyxin-derived deacylated nonapeptides which contain a heptapeptide ring and are known as effective permeabilizers of the outer membrane (OM) of Gram-negative bacteria. In order to assess the role of the cyclic moiety of PMBN in the permeabilization of the OM, the author compared the OM permeabilizing activity of two synthetic linear PMBN analogues with the well-characterized activity of PMBN. While a low concentration (1-3 micrograms/ml) of PMBN was sufficient to sensitize both Escherichia coli and Pseudomonas aeruginosa to hydrophobic probe antibiotics (rifampin, fusidic acid) by a factor of 100, even a high concentration (100 micrograms/ml) of linear arginyl polymyxin B decapeptide sensitized E. coli only by a factor of 3 and did not sensitize P. aeruginosa at all. In identical assays, linear lysyl polymyxin B nonapeptide completely lacked any sensitizing activity. These findings indicate that the cyclic peptide ring is crucial for the OM-permeabilizing activity of polymyxin nonapeptides.