One of the apparent roles of the outer membrane system in gram-negative bacteria is to function as a selective permeability barrier. A number of antibiotics active against gram-positive bacteria are relatively ineffective against gram-negative bacteria presumably because of the implied barrier function of the outer membrane. This interpretation has been strengthened by studies demonstrating synergism between outer membrane perturbing agents such as EDTA or polymyxin B and specific antibiotics. In the case of polymyxin B, it is not totally clear that synergism with other antimicrobials is due to disruption of the outer membrane permeability barrier or to interactions with the inner membrane. In order to resolve this question, polymyxin B was covalently attached to agarose in order to limit interactions with the outer surface of E. coli. These studies demonstrate that immobilized polymyxin B acts synergistically with bacitracin, rifampicin, or lysozyme. It is proposed that synergistic effects exhibited by polymyxin B are due to its interaction with the outer membrane system.