Previous reports have demonstrated that large cationic polypeptides (of molecular mass 5,000 daltons or greater) cause an increase in the apical membrane conductance of the rabbit urinary bladder epithelium. This report investigates the effects of the small cationic molecule polymyxin B (PX: a 1,400 dalton antibiotic) on the permeability of the rabbit urinary bladder. The addition of micromolar concentrations of polymyxin B to the luminal solution of the rabbit urinary bladder resulted in an increase in the transepithelial conductance of the bladder. The magnitude of the increase in the conductance was dependent upon the concentration of PX, and the polarity and magnitude of the apical membrane potential. As the apical membrane potential was made more cell interior negative, the larger was the increase in the membrane conductance. This voltage-dependent increase in conductance was an exponential function of the applied voltage, with a negligible increase in conductance occurring when the membrane potential was cell interior positive. Upon changing the membrane voltage from cell interior positive to negative, there was a delay before there was a measurable change in the membrane conductance. The longer the apical membrane was exposed to PX, the more poorly reversible was its effect on the transepithelial conductance, suggesting a toxic effect of PX on this epithelium.