This report demonstrates that Ca²⁺, Mg²⁺, and protons alter the ability of polymyxin B (PX, a cationic antibiotic used clinically as a bactericidal agent) to increase the apical membrane conductance of the rabbit urinary bladder. Using electrophysiological methods, we determine that these alterations occur by two mechanisms. First, they blocked the PX-induced conductance in a rapid and reversible manner; second, they competed with PX for a membrane binding site. In addition, Ca²⁺ (but not Mg²⁺ or protons) altered the rate at which the induced conductance could be reversed. When solution pH was greater than 8.8, PX was not able to induce a conductance. This ability of high pH to inhibit the action of PX was due to a decrease in the number of positive charges on PX. Further studies demonstrated that for maximal activity, PX required its fatty acid tail. These data were used to develop a model describing the mechanism by which PX can induce a conductance in the apical membrane of the rabbit urinary bladder.