Escherichia coli B cells were sensitized to ionophore A23187 by polymyxin B nonapeptide, and the induced magnesium and potassium ion fluxes were studied. Combined ionophore treatment permeabilized the cytoplasmic membrane of E. coli in an ion-specific manner and allowed the manipulation of intracellular Mg2+ content from the outside. A23187-induced Mg2+ efflux or influx was dependent on the free Mg2+ concentration gradient between the outside and inside of the cytoplasmic membrane and on the pH gradient. Most of the intracellular Mg2+ was bound, whereas only 1 to 2 mM was free in solution in the cellular sap.