During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.