Picosecond spectroscopy under a confocal microscope is employed to study solvation dynamics of coumarin 153 (C153) inside a single giant lipid vesicle (1,2-dilauroyl-sn-glycero-3-phosphocholine, DLPC) of diameter 20 μm. Fluorescence correlation spectroscopy (FCS) indicates that the diffusion coefficient (D t) of the probe (coumarin153, C153) in the immobilized vesicle displays a wide distribution from ∼3 to 21 μm 2 s -1. The distribution of D t suggests that the microenvironment of the probe (C153) is highly heterogeneous and the local friction is different for probe molecules in different regions. The values of D t is significantly smaller than that for the same dye in bulk water (550 μm 2 s -1). This suggests that the probe is located in the interface or membrane region rather than in the water pool of the vesicle. The solvation time of C153 in different regions of the lipid vesicle varies between 750 to 1200 ps. This result clearly shows that a confocal microscope is able to resolve the spatial heterogeneity in local friction (i.e., D t) and solvation dynamics within a lipid vesicle. © 2012 American Chemical Society.