The outer membrane-disorganizing effect of a short (10-min) treatment with polycationic agents was studied with smooth Salmonella typhimurium used as a test organism. The polycationic agents were the protamine salmine, a lysine polymer with 20 lysine residues (lysine20), and the deacylated polymyxin B derivative polymyxin B nonapeptide. Two different types of outer membrane-disorganizing were found. Protamine and lysine20 released 20 to 30% of the lipopolysaccharide from the outer membrane and sensitized the bacteria to the anionic detergent sodium dodecyl sulfate but did not (under these conditions) make the bacteria permeable to the hydrophobic probes fusidic acid and actinomycin D. In contrast, polymyxin B nonapeptide did not release lipopolysaccharide or sensitize the bacteria to sodium dodecyl sulfate but made the outer membrane permeable to the hydrophobic probes. None of the agents was bactericidal under the conditions used or caused any leakage of periplasmic beta-lactamase. Polymyxin B was used as a reference and showed characteristic outer membrane-disorganizing action. In thin-section electron microscopy, polymyxin B nonapeptide caused the appearance of long, narrow, finger-like projections on the outer membrane. Protamine and lysine20 caused a distinctly wrinkled appearance of the outer membrane but no projections.