The effect of channel-forming peptide gramicidin A on the dipole potential of phospholipid monolayers and bilayers has been studied. Surface pressure and surface potential isotherms of monolayers have been measured with a Langmuir trough equipped with a Wilhelmy balance and a surface potential meter (Kelvin probe). Gramicidin has been shown to shift pressure-area isotherms of phospholipids and to reduce their monolayer surface potentials. Both effects increase with the increase in gramicidin concentration and depend on the kind of phosphatidylcholine used. Application of the dual-wavelength ratiometric fluorescence method using the potential-sensitive dye RH421 has revealed that the addition of gramicidin A to dipalmitoylphosphatidylcholine liposomes leads to a decrease in the fluorescence ratio of RH421. This is similar to the effect of phloretin, which is known to decrease the dipole potential. The comparison of the concentration dependences of the fluorescence ratio for gramicidin and phloretin shows that gramicidin is as potent as phloretin in modifying the membrane dipole potential.