Hypothesis: Electrochemical impedance spectroscopy, EIS, can be used as a complementary technique to investigate ion interaction with the headgroup region in the aqueous channels of a lipid cubic phase, LCP. Experiments: A freestanding membrane made of monoolein LCP was formed by filling a small aperture that separates two cell compartments. The cell compartments were filled with electrolyte solutions at two different ionic strengths: i.e.: 10 and 100 mM, of KCl, CsBr and CaCl2. Electrochemical impedance spectroscopy was recorded between two platinum electrodes that were present at each side of the membrane. Findings: The membrane resistance and capacitance were estimated from equivalent circuit fitting of the impedance data. It was confirmed that calcium ions interacts strongly with the headgroup region in the aqueous channels giving significantly higher membrane resistances compared to monovalent alkali metal ions. The membrane capacitance with Ca2+(aq) in solution was concentration dependent, which for the first time indicates formation of two different cubic phases at these conditions. (C) 2016 Published by Elsevier Inc.