The mesoscopic organization adopted by two primary amphipathic peptides, P-(beta) and P-(alpha), in Langmuir-Blodgett (LB) films made of either the pure peptide or peptide-phospholipid mixtures was examined by atomic force microscopy. P-(beta), a potent cell-penetrating peptide (CPP), and P-(alpha), mainly differ by their conformational states, predominantly beta-sheet for P-(beta) and an alpha-helix for P-(alpha), as determined by Fourier transform infrared spectroscopy. LB films of pure peptide, transferred significantly below their collapse pressure, were characterized by the presence of supramolecular structures, globular aggregates for P-(beta) and filaments for P-(alpha), inserted into the monomolecular film. In mixed peptide-phospholipid films, similar structures could be observed, as a function of the phospholipid headgroup and acyl chain saturation. They often coexisted with a liquid-expanded phase composed of miscible peptide-lipid. These data strongly suggest that primary amphipathic CPP and antimicrobial peptides may share, to some extent, common mechanisms of interaction with membranes.