Defensins are small cationic antibacterial peptides that are abundant in polymorphonuclear leukocytes from human and other sources (T. Ganz, M. Selsted, D. Szklarek, S. Harwig, K. Daher, D. F. Bainton, and R. J. Lehrer, J. Clin. Invest. 76:1427-1435, 1985). We studied whether subinhibitory concentrations of defensins increase the outer membrane (OM) permeability of Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa to hydrophobic probes, as do many other polycations that have been studied previously. Throughout the study, we used polymyxin B nonapeptide (PMBN) as a reference peptide. PMBN has a known potent OM permeability-increasing action. As a sharp contrast to PMBN, subinhibitory concentrations of defensins did not permeabilize (or, under some test conditions, permeabilized very slightly) the OM to the probes that were used (rifampin and Triton X-100). At bacteriostatic or bactericidal defensin concentrations, some degree of synergism with rifampin was seen.