When human erythrocytes are treated with Ca2+ and ionophore A23187, two kinds of vesicles are shed from the surface, socalled microvesicles and nanovesicles. These vesicles were purified by differential centrifugation and their sizes and structures were determined by dynamic-force microscopy under physiological conditions. Microvesicles (Fig. 1a) and nanovesicles (Fig. 1b) were significantly different in size. Their size distributions showed maxima at 179 nm and 81 nm diameter, respectively. Higher magnification amplitude images resolved a patchy fine structure of the microvesicle membrane (Fig. 1c). Because microvesicles are free of cytoskeletal components, this fine structure apparently reflects the inherent domain organization of the vesicular membrane. Some of the microvesicles are associated with flat vesicular membrane domains (Fig. 1c), possibly indicating a domain segregation. Biochemical analysis revealed that microvesicles contain lipid rafts enriched in glycosyl phosphatidylinositol (GPI)-linked proteins, the lipid raft protein stomatin, and two cytosolic proteins, synexin and sorcin, which translocate to the membrane upon Ca2+ binding. Nanovesicles also contain lipid rafts, with synexin and sorcin being the most abundant proteins in the presence of Ca2+. Interestingly, whereas stomatin is specifically present in the microvesicular rafts, synexin and sorcin are present in microvesicular and nanovesicular rafts, the lipid raft proteins flotillin-1 and -2 are not found in the vesicles but remain in the red cell membrane. These data indicate the coexistence of different types of lipid rafts in the membrane and their segregation upon treatment with Ca2+. Synexin is suggested to be the driving force for the release of vesicles from erythrocytes.