Methyl-beta-cycloclextrin (M beta CD) can transfer phospholipids between vesicles, and its transfer ability has been utilized for the preparation of asymmetric vesicle and lipid incorporation into culture cells. Nevertheless, a detailed kinetic analysis of the M beta CD-mediated phospholipid transfer has not yet been carried out. We performed real-time monitoring of intervesicular lipid transfer by means of the fluorescence of pyrene-labeled phospholipids. Intermolecular excimer formation of the pyrenelabeled lipids in a membrane strongly depends on the local concentration of the fluorophore and decreases when the pyrenelabeled lipids are transferred froth donor (fluorophore,containing) vesicles to acceptor (fluorophore-free) vesicles. We monitored the fluorescence intensity of the pyrene monomer and excimer simultaneously and found that the excimer/monomer ratio decreased in the presence of M beta CD, pointing to M beta CD-mediated lipid transfer. The transfer rate depended on the M beta CD concentration but not on the lipid concentration, suggesting that dissociation from the membrane via extraction by M beta CD is the rate-limiting step of the lipid transfer. Calibration of the excimer/monomer ratio to the molar fraction of the pyrene-labeled lipids enabled us to evaluate the dissociation rate constant correctly. From the temperature dependence of the transfer, we obtained the thermodynamic activation parameters, which revealed that the extraction of phosphatidylcholine by M beta CD from membranes is less enthalpically unfavorable than that of phosphatidylglycerol and phosphatidylinositol.