The interaction of amphiphilic helical peptides with phospholipid membranes of various diameters was studied. H-(Leu-Aib-Lys-Aib-Aib-Lys-Aib)(3)-OCH3 (P21OMe) was bound to large unilamellar vesicles (LUV) of 100 nm and larger diameter or to small unilamellar vesicles (SUV) of dimyristoylphosphatidylcholine (DMPC). The helix content of P21OMe increased remarkably upon partition to phospholipid bilayer membrane. A short peptide, H-Leu-Aib-Lys-Aib-Lys-Aib-OBzl (P7Bz), did not take helical conformation, and was not bound to any type of vesicle. Therefore, the amphiphilic helical structure is a key factor for peptide binding to phospholipid bilayer membranes. Two octadecyl chains at the C terminus of a short peptide, H-Leu-Aib-Lys-Aib-Aib-Lys-Aib-Ala-N (C18H37)(2), increase the affinity to the phospholipid membrane. On the other hand, the connection of two octadecyl chains to P21OMe, H-(Leu-Aib-Lys-Aib-Aib-Lys-Aib)(3)-Ala-N (C18H37)(2), increase the affinity to the phospholipid membrane. On the other hand, the connection of two octadecyl chains to P21OMe, H-(Leu-Aib-Lys-Aib-Aib-Lys-Aib)(3)-Ala-N (C18H37)(2) (P21D), prevents the peptide from binding to LUV of diameters larger than 100 nm, while it is partitioned to SUV. It was thus shown that the effect of two octadecyl chains on the peptide binding to phospholipid bilayer membranes changes, depending on the peptide and the size of phospholipid vesicles.