Antimicrobial peptide magainin 2 (mag) forms pores in lipid membranes and induces membrane permeation of the cellular contents. Although this permeation is the likely main cause of its bactericidal activity, the mechanism of pore formation remains poorly understood. We therefore investigated in detail the interaction of mag with lipid membranes using single giant unilamellar vesicles (GUVs). The binding of mag to the lipid membrane of GUVs increased the fractional change in the area of the membrane, δ, which was proportional to the surface concentration of mag, X. This indicates that the rate constant of mag-induced pore formation greatly increased with an increase in δ. The tension of a lipid membrane following aspiration of a GUV also activates mag-induced pore formation. These results show that the stretch of the lipid membrane is a main driving force of mag-induced pore formation, and the rate constant of mag-induced pore formationgreatly increases as the stretch of the lipid membrane increases, indicating that the mag-induced pore is a stretch-activated pore. Simultaneous measurements of the leakage of a fluorescent probe and the location of carboxyfluorescein (CF)-labeled mag (CF-mag), and simultaneous measurements of the time course of the CF-mag-induced change in the area of the GUV membrane and the CF-mag concentration in the GUV membrane, together strongly indicate that mag cannot translocate from the outer to the inner monolayer until just before pore formation. Based on these results, we discuss the possible mechanism for mag-induced pore formation.