The mechanisms of six different antimicrobial, cytolytic, and cell-penetrating peptides, including some of their variants, are discussed and compared. The specificity of these polypeptides varies; however, they all form amphipathic alpha-helices when bound to membranes, and there are no striking differences in their sequences. We have examined the thermodynamics and kinetics of their interaction with phospholipid vesicles, namely, binding and peptide-induced dye efflux. The thermodynamics of binding calculated using the Wimley-White interfacial hydrophobicity scale are in good agreement with the values derived from experiment. The generally accepted view that binding affinity determines functional specificity is also supported by experiments in model membranes. We now propose the hypothesis that it is the thermodynamics of the insertion of the peptide into the membrane, from a surface-bound state, that determine the mechanism.