The interaction of alamethicin with lecithin bilayer vesicles in the presence of a transmembrane potential difference was studied by PMR spectroscopy. Asymmetrical vesicles with alamethicin trapped within the intravesicular compartment were prepared, and the formation of ion channels was then triggered by imposing a suitable K+ gradient across the bilayer membrane in the presence of valinomycin. These alamethicin channels permitted the outward passage of Eu ions into the extravesicular medium when the transmembrane potential is more positive for the inner compartment.