Cytosolic Ca(++) induces the shedding of microvesicles and nanovesicles from erythrocytes. Atomic force microscopy was used to determine the sizes of these vesicles and to resolve the patchy, fine structure of the microvesicle membrane. The vesicles are highly enriched in glycosyl phosphatidylinositol-linked proteins, free of cytoskeletal components, and depleted of the major transmembrane proteins. Both types of vesicles contain 2 as-yet-unrecognized red cell proteins, synexin and sorcin, which translocate from the cytosol to the membrane upon Ca(++) binding. In nanovesicles, synexin and sorcin are the most abundant proteins after hemoglobin. In contrast, the microvesicles are highly enriched in stomatin. The membranes of both microvesicles and nanovesicles contain lipid rafts. Stomatin is the major protein of the microvesicular lipid rafts, whereas synexin and sorcin represent the major proteins of the nanovesicular rafts in the presence of Ca(++). Interestingly, the raft proteins flotillin-1 and flotillin-2 are not found in the vesicles but remain in the red cell membrane. These data indicate the presence of different types of lipid rafts in the erythrocyte membrane with distinct fates after Ca(++) entry. Synexin, which is known to be vital to the process of membrane fusion, is suggested to be a key component in the process of vesicle release from erythrocytes.