The HNP-1, HNP-2, and HNP-3 defensins are human antimicrobial peptides produced in response to microbial invasion. Their properties are distinct, with a more potent action for HNP-3. In this study, the relationship between their structural dissimilarities and their different microbial actions was evaluated by molecular dynamics simulation. Structural determinants related to their intra- and intermolecular interactions were defined for each HNP using a simplified membrane model consisting of a water/n-hexane interface. The hydrophobic portion of the HNPs promotes their diffusion to the interface with a concomitant, slight change in the structure induced by the intermolecular electrostatic interactions between the HPN molecules and the interface. As a consequence, different orientations are probably adopted by the HNPs at the interface, which may explain their different actions. The interaction of HNP-1 and HNP-2 with the surfaces was also studied using Langmuir monolayers as a biomimetic system. It was found that peptides adsorb rapidly at n-hexane/water interfaces as well as at phospholipid Langmuir monolayers but not at the air/liquid interface. This reveals that the presence of an organic phase is required for the exposure of the hydrophobic groups of the peptides. In addition, adsorption kinetics and surface pressure-area isotherms for Langmuir monolayers suggested that the lipidpeptide interaction is strongly influenced by the monolayer electrical charge and packing, depending also on the HPN structure. This study supports a model in which defensins, acting in a dimeric form, are able to disrupt membranes. The model also shows that the individual structures of the HNPs are responsible for their different actions on microbes.