Cardiolipin (CL) lipids are present in the inner membrane of Gram-negative and Gram-positive bacteria as well as in the one of mithocondria and chloroplasts. The unique structure of this class of lipids is responsible for peculiar physical properties. NR is a well-established technique that can be used to obtain unique and detailed information on lipid bilayers. Through the evaluation of the scattering length density profile, a clear picture of the organization of cardiolipin in lipid bilayers in the presence and in the absence of divalent ions (Ca2+) can be produced. These results will complete the characterization of the impact of CL on the organization of phospholipids. We believe that the collected results will contribute to enlarge the state-of-the-art of CL-containing lipid systems and will represent a useful reference for those who are interested in characterizing biological processes, in which CL is involved, i.e. interaction with proteins.