Hypothesis: Electrochemical Impedance Spectroscopy (EIS) can be used to investigate cationic interaction with the choline headgroup in the ternary system of monoolein/dioleoylphosphatidylcholine/water (MO/DOPC/H2O). Experiments: EIS was used to estimate the resistance and capacitance of a freestanding membrane of a lipid cubic phase (LCP). The membrane was formed in a small cylindrical aperture separating two compartments, containing one Pt electrode each. The impedance experiments were carried out in a two electrode setup with electrolyte solutions made of KCl, CsCl, MgCl2 and CaCl2 filling the compartments at two different ionic strength. Small angle X-ray diffraction (SAXRD) was used to establish the structure and cell unit parameters of the LCP. Findings: The interpretation of ionic interaction with phosphatidylcholine was based on estimated membrane resistances and capacitances from EIS measurements. The magnitude of cationic interaction with the lipid headgroup in the water channels is correlated to the membrane resistance that increases in the order Cs+ < K+ < Mg2+ < Ca2+ following the Hofmeister direct series and also reflecting the order of intrinsic binding constants. The membrane capacitance and SAXRD results are discussed as an effect of cationic interaction and it was possible to observe both swelling and condensing effects. The stability of the cubic phase throughout the experiments was confirmed by SAXRD. (C) 2018 Elsevier Inc. All rights reserved.