The permeation of water molccules across single-component lecithin or lecithin-cholesterol bilayers is studied by a new technique. The new technique makes use of the different fluorescence quantum yields of appropriate molecules in D2O and H2O. Water-soluble indole derivatives which by experimental manipulation reside almost entirely within the aqueous (H2O) intravesicular compartment thus can monitor D2O molecules permeating the bilayer by virtue of an increased quantum yield of the fluorescence. In a stopped-flow instrument, a vesicle solution containing the fluorescent chromophore in the intravesicular space is rapidly mixed with the deuterated solvent. The approach to the “steady state”, where the intra- and extravesicular D2O and H2O concentrations are equal, proceeds in a single-exponential manner. Consequently, the exchange relaxation time for the D2O molecules passing the bilayer can be deduced from the time-dependent increase of the fluorescence intensity. The method and results on lecithin and lecithin-cholesterol bilayer vesicles are discussed. The exchange relaxation times of temperature-dependent studies are interpreted within the framework of the solubility-diffusion theory. Below the crystalline to liquid-crystalline phase transition temperature and for cholesterol-free vesicles, the rate-limiting step for the D2O permeation is attributed to the intracore diffusion. Above the phase transition and for cholesterol-containing vesicles, the intracore diffusion seems not to be rate-limiting. Deviations from the linearity below the phase transition in the Arrhenius-type presentation of the data are related to changes of the partition coefficient of water between the solvent and the lipid phase at the premelting temperature.