The pore-forming bacterial toxins of the colicin family undergo massive protein refolding, while attacking a target cell, to convert from the water-soluble conformational state to the membrane-associated state with subsequent insertion of helical hairpins into the cytoplasmic membrane. To explore the validity of proposed models for the mechanism by which the soluble channel-forming domain of colicin A turns inside out upon membrane association, five site-specific cysteine mutants of colicin A, each singly spin labeled with nitroxide side chains, were studied by 9.5 GHz (X-band) and 95 GHz (W-band) high-field EPR. By elucidating the mobility of the nitroxide side chains on one of the two hydrophobic helices and their accessibility to paramagnetic relaxer molecules in the membrane, as well as by measuring the g(xx), and A(zz) nitroxide tensor components, detailed information about conformational changes upon membrane association could be revealed. This information on the channel-forming domain of colicin A goes beyond that available already from X-ray crystallography. The multifrequency EPR results are in favor of the "penknife" model of the membrane-associated channel-forming domain of colicin A with the ion channel still closed in the absence of additional modulation of the membrane potential. The results cannot exclude the existence of a thermodynamic equilibrium with other conformations in which the "penknife" state is predominantly populated.