In contrast to their polymyxin-susceptible parent strains, polymyxin-resistant Salmonella typhimurium mutants (pmrA strains) did not lose their outer membrane permeability barrier to macromolecules such as lysozyme and periplasmic proteins upon polymyxin treatment. The sensitization of pmrA strains to deoxycholate-induced lysis required 10-times-higher polymyxin concentrations than did the sensitization of the parent strains. These findings indicate that the pmrA mutation affects the outer membrane and decreases its susceptibility to polymyxin. By contrast, the pmrA mutants did not differ from their parents in the uptake of gentian violet after treatment with polymyxin, suggesting a degree of specificity in the pmrA effect in the outer membrane.