The eosinophil granule protein major basic protein (MBP) is toxic to a wide variety of cell types, by a poorly understood mechanism. To determine whether the action of MBP involves an alteration in membrane permeability, we tested purified MBP on rabbit urinary bladder epithelium using transepithelial voltage-clamp techniques. Addition of nanomolar concentrations of MBP to the mucosal solution caused an increase in apical membrane conductance only when the voltage across the apical membrane was cell interior negative. The magnitude of the MBP-induced conductance was a function of MBP concentration, and the rate of the initial increase in conductance was a function of the transepithelial voltage. The MBP-induced conductance was nonselective for K⁺ and Cl. Mucosal Ca²⁺ reversed the induced conductance, whereas mucosal Mg²⁺ partially blocked the induced conductance and slowed the rate of the increase in conductance. The induced conductance was partially reversed by changing the voltage gradient across the apical membrane to cell interior positive. Prolonged exposure resulted in an irreversible loss of the barrier function of the urinary bladder epithelium. These results suggest that an increase in cell membrane ion permeability is an initial step in MBP-induced loss of barrier function.