Membrane lysis caused by antibiotic peptides is often rationalized by means of two different models: the so-called carpet model and the pore-forming model. We report the lytic activity of antibiotic peptides from Australian tree frogs: Maculatin 1.1, Citropin 1.1 and Aurein 1.2, on palmitoyloleoylposphatidylcholine (POPC) or POPC/POPG (phosphatidylglycerol) model membranes. Leakage experiments using fluorescence spectroscopy indicated that the peptide to lipid mole ratio necessary to induce a 50% of probe leakage was smaller for Maculatin compared with Aurein or Citropin, regardless of lipid membrane composition. To gain further insight into the lytic mechanism of these peptides we performed single vesicle experiments using confocal fluorescence microscopy. In these experiments, the time-course of leakage for different molecular weight (water soluble) fluorescent markers incorporated inside single giant unilamellar vesicles is observed after peptide exposure. We conclude that Maculatin and its related peptides demonstrate a pore-forming mechanism (differential leakage of small fluorescent probe compared with high molecular weight marker). Conversely, Citropin and Aurein provoke a total membrane destabilization with vesicle burst without sequential probe leakage, an effect that can be assigned to a carpeting mechanism of lytic action. Additionally, in order to study the relevance of the proline residue on the membrane-action properties of Maculatin, the same experimental approach was used for Maculatin-Ala and Maculatin-Gly (Pro-15 was replaced by Ala or Gly, respectively). Although a similar peptide to lipid mole ratio was necessary to induce 50% of leakage for POPC membranes, the lytic activity of Maculatin-Ala and Maculatin-Gly decreased in POPC/POPG (1:1 mol) membranes compared with that observed for the naturally occurring Maculatin sequence. As observed for Maculatin, the lytic action of Maculatin-Ala and Maculatin-Gly is in keeping with the formation of pore-like structures at the membrane independently of lipid composition.