The effects of polymyxin B (PMB) on the Escherichia coli outer (OM) and cytoplasmic membrane (CM) permeabilities were studied by monitoring the fluxes of tetraphenylphosphonium, phenyldicarbaundecaborane, and K(+) and H(+) ions. At concentrations between 2 and 20 microgram/ml, PMB increased the OM permeability to lipophilic compounds and induced a leakage of K(+) from the cytosol and an accumulation of lipophilic anions in the cellular membranes but did not cause the depolarization of the CM. At higher concentrations, PMB depolarized the CM, forming ion-permeable pores in the cell envelope. The permeability characteristics of PMB-induced pores mimic those of bacteriophage- and/or bacteriocin-induced channels. However, the bactericidal effect of PMB took place at concentrations below 20 microgram/ml, indicating that this effect is not caused by pore formation. Under conditions of increased ionic strength, PMB made the OM permeable to lipophilic compounds and decreased the K(+) gradient but was not able to depolarize the cells. The OM-permeabilizing effect of PMB can be diminished by increasing the concentration of Mg(2+). The major new findings of this work are as follows: (i) the OM-permeabilizing action of PMB was dissected from its depolarizing effect on the CM, (ii) the PMB-induced ion-permeable pores in bacterial envelope were registered, and (iii) the pore formation and depolarization of the CM are not obligatory for the bactericidal action of PMB and dissipation of the K(+) gradient on the CM.