The activity of PGLa, an antimicrobial peptide isolated from hemocytes of frog skin and its secretions on living Escherichia coli, was investigated by imaging the cells with atomic force microscopy (AFM) in physiological conditions and by measuring its cellular stiffness. The treatment of bacteria with the antimicrobial peptide PGLa in the culture medium had two stages. The first was characterized by the loss of surface stiffness and consequent loss of bacteria topographic features and the formation of micelles probably originating from the disruption of the outer membrane. The formation of outer membrane originated micelles is in agreement with the carpet-like mechanism of action proposed for antimicrobial peptides of the magainin family. The peptide action also resulted in the removal of bacterial pili. In a second stage there was further damage which resulted in total cell rupture. The addition of Mg(2+) ions prior to peptide treatment partially inhibited the effects of PGLa on bacteria. This result suggests that PGLa interacts with the outer membrane by displacing Mg(2+) from LPS, inserting itself into the bilayer and cross-bridging the negative charges of LPS lipids as proposed in the self-promoted pathway mechanism. The peptide effect on the bacteria was compared to the activity of the chelating agent EDTA that damages the bacterial outer membrane by removing Mg(2+) ions.