Cell penetrating peptides are promising vectors for molecular drug delivery in eukaryotic cells. Despite of their discovery 20 years ago, the mechanisms of peptide membrane crossing are still controversial. The different suggested penetration mechanisms reflect the high sequence and structural diversity of cell penetrating peptides. The fundamental step for peptide penetration into the cytosol is the crossing of the membrane lipid barrier at the level of the plasma membrane or the endosomes. Therefore, the study of the peptide-lipid interaction is the key for peptide penetration mechanisms understanding. In order to study the changes in lipid organisation induced by the cell penetrating peptide penetratin, several experiments by three different physicochemical approaches were performed. X-ray diffraction data shows that penetratin is able to induce membrane phase separation and lipid rearrangements observed by inter-lipid distances. These changes are accompanied by a temperature stable behaviour of some of the induced membrane domains. The membrane environment fluorescent probe laurdan showed that, in DMPC and DMPC/DMPG membranes, the peptide induces de-packing of lipids. Calorimetric analyses show that penetratin favours the gel phase to gel-like rippled phase transition. Overall, the data suggest both, that the rippled phase is a heterogeneous structure formed by gel-like and fluid-like coexisting components, and that the penetratin-induced membrane heterogeneity could be important for membrane destabilisation during cell penetration.