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 similar to 100 nm) is smaller than the diffraction limited spot size (similar to 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 degrees C, respectively) were studied. The diffusion coefficient of the dye DCM in bulk water is similar to 300 mu m(2)/s. In the lipid vesicle, the average D-t decreases markedly to similar to 5 mu m(2)/s (similar to 60 times) in the gel phase (for DPPC at 20 degrees C) and 40 mu m(2)/s (similar to 8 times) in the fluid phase (for DLPC at 20 degrees C). This clearly demonstrates higher mobility in the fluid phase compared with the gel phase of a lipid. It is observed that the D-t values vary from lipid to lipid and there is a distribution of D-t values. The diffusion of the hydrophobic dye DCM (D-t similar to 5 mu m(2)/s) in the DPPC vesicle is found to be 8 times smaller than that of a hydrophilic anioinic dye C343 (D-t similar to 40 mu m(2)/s). This is attributed to different locations of the hydrophobic (DCM) and hydrophilic (C343) dyes.