The PhoPQ two-component system of Salmonella enterica serovar Typhimurium produces a remodeling of the lipid A domain of the lipopolysaccharide, including the PagP-catalyzed addition of palmitoyl residue, the PmrAB-regulated addition of the cationic sugar 4-aminoarabinose and phosphoethanolamine, and the LpxO-catalyzed addition of a 2-OH group onto one of the fatty acids. By using the diffusion rates of the dyes ethidium, Nile red, and eosin Y across the outer membrane, as well as the susceptibility of cells to large, lipophilic agents, we evaluated the function of this membrane as a permeability barrier. We found that the remodeling process in PhoP-constitutive strains produces an outer membrane that serves as a very effective permeability barrier in an environment that is poor in divalent cations or that contains cationic peptides, whereas its absence in phoP null mutants produces an outer membrane severely compromised in its barrier function under these conditions. Removing combinations of the lipid A-remodeling functions from a PhoP-constitutive strain showed that the known modification reactions explain a major part of the PhoPQ-regulated changes in permeability. We believe that the increased barrier property of the remodeled bilayer is important in making the pathogen more resistant to the stresses that it encounters in the host, including attack by the cationic antimicrobial peptides. On the other hand, drug-induced killing assays suggest that the outer membrane containing unmodified lipid A may serve as a better barrier in the presence of high concentrations (e.g., 5 mM) of Mg(2+).