Fluorescence correlation spectroscopy (FCS) has been used to study the motion of fluorescent dyes in a giant (diameter 20 000 nm = 20 μm) catanionic vesicle comprised of the surfactant sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide (DTAB). The diffusion in the anion (SDS) rich catanionic vesicle was studied both in bulk water and in an immobilized vesicle attached to a positively charged glass surface. In the case of the immobilized vesicle, the diffusion coefficients (Dt) of R6G (rhodamine 6G), DCM (4-dicyanomethylene-2-methyl-6-p-dimethyl aminostyryl-4H-pyran), and C343 (coumarin 343) are found to be 1.5, 2.5, and 10 μm2/s, respectively, which are 280, 120, and 55 times slower compared to those for the same dyes in bulk water. The magnitude of Dt is found to vary for different vesicles. This was attributed to the difference in size and shape of the immobilized vesicles. In bulk, R6G binds completely to the vesicle and exhibits extremely slow diffusion with Dt = 0.5 ± 0.1 μm2/s (∼850 and 3 times slower compared to that of R6G in bulk water and within the immobilized vesicle). This is attributed to very slow overall diffusion of the very large size vesicles (20 μm = 20 000 nm). Both of the dye molecules (DCM and C343) show two different diffusion coefficients for the vesicles in bulk. In this case, the small Dt (0.5 ± 0.1 μm2/s) corresponds to the diffusion of the vesicle as a whole and the large Dt value (300 and 550 μm2/s for DCM and C343, respectively) corresponds to the free dye molecules in bulk water. © 2010 American Chemical Society.