The membrane-disrupting properties of cecropin A (1-8)-magainin 2 (1-12) hybrid peptide, which has higher antitumor with less hemolytic activities than cecropin A (1-8)-melittin (1-12), and its analogues were assessed by measuring the induced release of vesicle-entrapped fluorescence probes. A model membrane was made of zwitterionic phospholipid (phosphatidylcholine) or the mixture of negatively and zwitterionic phospholipids (phosphatidylcholine and phosphatidylserine). The extent of leakage of the aqueous content of the phospholipid vesicles was found to have remarkable discrepancies according to the amphipathic nature of each analogue peptide. The entrapped high molecular weight solute (fluorescein-labeled immunoglobulin G, 55 kDa) also was released by the analogue which had the largest hydrophobic region and the highest amphipathic score among peptides tested. As the result of the determination of the relationships between the membrane-disrupting properties and the hydrophobicity values of peptides, it was found that the membrane-disrupting activity increased according to increasing the hydrophobicity of the peptide. The tryptophan fluorescence emission spectra and CD spectra showed that on interaction with the phospholipid vesicle, the peptide acquired the ordered structure and alpha-helical conformation by moving a tryptophan residue into the nonpolar environment of the phospholipid vesicle. These results suggest that the breakdown of the lipid bilayer was mediated by the alpha-helical amphipathic structure of the peptide interacting with the lipid bilayers as well as the by the hydrophobicity of the peptide.