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Interactions of antimicrobial peptides with lipid membranes have been investigated using a suspension of small liposomes, and their details remain unclear. Using the single GUV method, we have succeeded in revealing elementary processes of the pore formation in lipid membranes induced by magainin 2 in our previous paper (Biochemistry, 44, 15823, 2005). In this report, to elucidate the mechanism of the magainin 2-induced pore formation, we investigated the effect of surface charge density of membranes on the pore formation. To change the surface charge density, we controlled negatively charged DOPG concentration in DOPG/DOPC membrane from 30 to 60 mol%. We found that, in all kinds of GUVs, magainin 2 induced a rapid leakage of calcein from single GUVs, showing that magainin 2 formed pores in the membrane. For GUVs with the same charge density, the fraction of leaked GUV, PLS, increased with time, and PLS at a fixed time increased with magainin 2 concentration. The magainin 2 concentration at PLS = 0.5 at a fixed time increased with a decrease in the surface charge density, indicating that higher concentrations of magainin 2 in a buffer were required to induce the pore formation in GUVs with lower surface charge density. Using the Gouy-Chapman theory, we obtained magainin 2 concentration in the membrane interface, assuming the intrinsic binding constant of magainin 2 with DOPG/DOPC membranes. On the basis of these results, we discuss the role of magainin 2 concentration in the membrane interface in the magainin 2-induced pore formation.