The dipole potential of a lipid bilayer membrane accounts for its much larger permeability to anions than cations and affects the conformation and function of membrane proteins. The absolute value of the dipole potential has been very difficult to measure, although its value has been estimated to range from 200 to 1,000 mV from ion translocation rates, the surface potential of lipid monolayers, and molecular dynamics calculations. Here, a point charge probe method was used to investigate the dipole potentials of both ester and ether lipid membranes. The interactions between electrons and lipid molecules were recorded by phase-contrast imaging using cryo-EM. The magnitude and the profile of the dipole potential along the bilayer normal were obtained by subtracting the contribution of the atomic potential from the cryo-EM image intensity. The peak dipole potential was estimated to be 510 and 260 mV for diphytanoylphosphatidylcholine and diphytanylphosphatidylcholine, respectively.