This paper describes the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabiit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increases the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential the larger the conductance increase. The concentration dependence of the conductance increase saturated suggesting a membrane-binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites, one was competition with colistin for a membrane binding site, the other was by rapidly blocking the induced conductance. Short exposure times allowed the increase in conductance to be reversed by either colistin removal from the bath, or by changing the voltage so that the apical membrane was cell interior positive. Long exposure times caused the conductance increase to be only partially reversible by colistin removal from the bath or by changing the voltage to cell interior positive. This suggests that at long exposure times there is a toxic effect of colistin on the urothelium.