The pore formation in lipid membranes induced by the antimicrobial peptide magainin 2 is considered to be the main cause for its bactericidal activity. To reveal the mechanism of the pore formation, it is important to elucidate the kinetic pathway of magainin 2-induced pore formation in lipid membranes. In this report, to examine the change in pore size over time during pore formation which can monitor its kinetic pathway, we investigated the rate of the leakage of various sized fluorescent probes through the magainin 2-induced pores in single giant unilamellar vesicles (GUVs) of 50% dioleoylphosphatidylglycerol (DOPG)/50% dioleoylphosphatidylcholine (DOPC) membrane. Magainin 2-induced leakage of Texas-Red dextran 10000, Texas-Red dextran 3000, and Alexa-Fluor trypsin inhibitor occurred in two stages; a transient rapid leakage in the initial stage followed by a stage of slow leakage. In contrast, magainin 2 induced a transient, but very small (10-20%), leakage of fluorescent probes of a larger size such as Texas-Red dextran 40 000 and FITC-BSA. These results indicate that magainin 2 molecules initially induce a large, transient pore in lipid membranes following which the radius of the pore decreases to a stable smaller size. We estimated the radius of these pores, which increases with an increase in magainin 2 concentration. On the basis of these data, we propose a hypothesis on the mechanism of magainin 2-induced pore formation.