The effect of glycerol on the permeability of vesicle membranes of a siloxane surfactant, the block copolymer polyethyleneoxide-b-polydimethylsiloxane-polyethyleneoxide, (EO)(15)-(DMS)(15)-(EO)(15), was studied with freeze-fracture transmission electron microscopy (FF-TEM) and pulsed-field gradient nuclear magnetic resonance (PFG-NMR) spectroscopy. The FF-TEM results show that, in pure water, the surfactant can form small vesicles with diameters of less than 25 nm, as well as a few multilamellar vesicles with diameters larger than 250 nm. Gradual substitution of water with glycerol to a glycerol content of 40% leads to significant structural transformations: small vesicles are gradually swollen, and large multilamellar vesicles disappear. A glycerol content of 60% results in the complete disintegration of the vesicles into membrane fragments. PFG-NMR measurements indicate that the vesicle membrane does not represent an effective barrier for water molecules on the NMR time scale; hence, the average residence time of water in the encapsulated state is below tau(b) = 2 ms. In contrast, the average residence time of glycerol molecules in the encapsulated state can be as large as tau(b) = 910 ms. The permeability of the vesicle membrane increases with increasing glycerol concentration in the solvent: At a concentration of 40%, the residence time tau(b) is lowered to approximately 290 ms. After vesicle destruction at higher glycerol concentrations, a small glycerol fraction is still bound by membrane fragments that are formed after the disintegration of the vesicles.