Antimicrobial peptides are innate host defense molecules with the ability to kill pathogens. They have been widely studied for their membrane lytic activity and their potential to overcome the ever-increasing threat of antimicrobial resistance against conventional antibiotics. Here, we focus on two halictines, antimicrobial peptides first obtained from the venom of the eusocial bee Halictus sexcinctus. The peptides, HAL-1 and HAL-2, are cationic (with +3 and +4 charges, respectively) and amphipathic, have 12 amino acid residues, and exhibit high biological activity. For this study, the mechanism of action of HAL-1 and HAL-2 was studied in detail using large and giant unilamellar vesicles composed of pure palmitoyl oleoyl phosphatidyl choline (POPC) and a mixture of POPC and the anionic lipid palmitoyl oleoyl phosphatidyl glycerol (POPG) as biomimetic models of the membranes of eukaryotes and microorganisms, respectively. A set of complementary techniques was put forward: carboxyfluorescein leakage assay, phase contrast optical microscopy, zeta-potential, static and dynamic light scattering, fluorescence and circular dichroism spectroscopies, and isothermal titration calorimetry. The results show that both halictines are able to interact strongly with anionic membranes: The interaction is exothermic and accompanied by structuring of the peptides as an alpha-helix and deep insertion into the membrane causing substantial membrane permeabilization at very low peptide/lipid molar ratios. Extensive vesicle aggregation was detected only at a high peptide concentration. On the other hand, the interaction of the halictines with POPC is significantly milder. Yet, the peptides were able to permeabilize the POPC membranes to some extent. Comparing both peptides, HAL-1 showed a somewhat stronger effect on model membranes. Fits to the data revealed apparent binding constants on the order of 10(3)-10(4) M-1 for anionic membranes and 1 order of magnitude lower for zwitterionic bilayers. When lytic activity results were compared at the same bound peptide/lipid ratio, the halictines exhibited a higher activity toward zwitterionic membranes. As novel peptides, small and with powerful activity, these halictines are potential candidates for becoming antimicrobial agents.