Synthetic fluorescent analogs of the natural lipopeptide trichogin GA IV were used to investigate the peptide position and orientation in model membranes. A translocation assay based on Forster energy transfer indicates that trichogin is associated to both the outer and inner leaflet of the membrane, even at low concentration, when it is not active. Fluorescence quenching measurements, performed by using water soluble quenchers and quenchers positioned in the membrane at different depths, indicate that at low membrane-bound peptide/lipid ratios trichogin lies close to the region of polar headgroups. By increasing peptide concentration until membrane leakage takes place, a cooperative transition occurs and a significant fraction of the peptide becomes deeply buried into the bilayer. Remarkably, this change in peptide position is strictly coupled with peptide aggregation. Therefore, the mechanism of trichogin action can be envisaged as based on a two-state transition controlled by peptide concentration. One state is the monomeric, surface bound and inactive peptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity.