The mobility of the organic dye DCM (4-dicyanomethylene-2-methyl-6-p- dimethyl aminostyryl-4H-pyran) in the gel and fluid phases of a lipid vesicle is studied by fluorescence correlation spectroscopy (FCS). Using FCS, translational diffusion of DCM is determined in the gel phase and fluid phase of a single lipid vesicle adhered to a glass surface. The size of a lipid vesicle (average diameter ∼100 nm) is smaller than the diffraction limited spot size (∼250 nm) of the microscope. Thus, the vesicle is confined within the laser focus. Three lipid vesicles (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-dipalmitoyl-sn- glycero-3-phosphocholine (DPPC)) having different gel transition temperatures (-1, 23, and 41 °C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is ∼300 μm2/s. In the lipid vesicle, the average Dt decreases markedly to ∼5 μm 2/s (∼60 times) in the gel phase (for DPPC at 20 °C) and 40 μm2/s (∼8 times) in the fluid phase (for DLPC at 20 °C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the Dt values vary from lipid to lipid and there is a distribution of Dt values. The diffusion of the hydrophobic dye DCM (Dt ∼ 5 μm2/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (Dt ∼ 40 μm2/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes. © 2010 American Chemical Society.