The study investigates the dynamics and size of lipid rafts in living mammalian cells by measuring the local diffusion of single membrane proteins. Using a laser trap to confine a bead bound to a raft protein, the researchers measured its local diffusion with high resolution single particle tracking. The results show that the method provides accurate measurements of the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins are raft-associated, their diffusion is independent of the type of membrane anchor and is significantly reduced compared to non-raft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins, with GPI-anchored proteins diffusing even faster than transmembrane proteins. GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 minutes. The measurements agree with the hypothesis that lipid rafts are cholesterol-stabilized complexes of 26 ± 13 nm in size, diffusing as one entity for minutes. The study provides insights into the stability and mobility of lipid rafts, supporting the model of cholesterol-stabilized rafts associating with proteins in the native state of the cell membrane.The study investigates the dynamics and size of lipid rafts in living mammalian cells by measuring the local diffusion of single membrane proteins. Using a laser trap to confine a bead bound to a raft protein, the researchers measured its local diffusion with high resolution single particle tracking. The results show that the method provides accurate measurements of the viscous damping of the membrane domain in the lipid bilayer. When glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins are raft-associated, their diffusion is independent of the type of membrane anchor and is significantly reduced compared to non-raft transmembrane proteins. Cholesterol depletion accelerates the diffusion of raft-associated proteins, with GPI-anchored proteins diffusing even faster than transmembrane proteins. GPI-anchored proteins were never observed to dissociate from the raft within the measurement intervals of up to 10 minutes. The measurements agree with the hypothesis that lipid rafts are cholesterol-stabilized complexes of 26 ± 13 nm in size, diffusing as one entity for minutes. The study provides insights into the stability and mobility of lipid rafts, supporting the model of cholesterol-stabilized rafts associating with proteins in the native state of the cell membrane.