In this work the presence of inverted hexagonal phases HII of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca2+ were observed via 31P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, HII phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being âˆ¼ 1Â nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to HII phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (âˆ¼ 100Â nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from HII phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.