The exclusively gram-positive antibacterial spectrum of the lipopeptide daptomycin (LY146032) suggests that the underlying basis for intrinsic resistance in gram-negative organisms involves envelope impermeability. This study was undertaken to determine whether the outer membranes of Pseudomonas aeruginosa and Pasteurella multocida can be rendered permeable to daptomycin by experimental modifications that result in susceptibility of gram-negative bacteria to lipophilic molecules. Turbidimetric growth assays revealed sublethal concentrations of polymyxin B or ethylenediaminetetraacetate (EDTA) sensitized all strains examined to the hydrophobic antibiotic novobiocin. Neither permeabilizer rendered Ps. aeruginosa or a hydrophilic Pa. multocida variant susceptible to daptomycin; however, polymyxin B sensitized a hydrophobic Pa. multocida variant, whereas EDTA did not. Cells cultured with sublethal concentrations of polymyxin B or EDTA retained negatively charged cell surfaces comparable to those of control cells. Growth of Pa. multocida strains in the presence of polymyxin B did not result in modification of cell envelope lipid composition. These findings indicate that the ability of the outer membrane to retard the diffusion of daptomycin does not require normally intact structure, thereby suggesting that the residual negative charge of the cell surface may preclude interaction with the acidic antibiotic owing to electrostatic repulsion.