Exosomes are 60 -100-nm membrane vesicles that are secreted into the extracellular milieu as a consequence of multivesicular body fusion with the plasma membrane. Here we determined the protein and lipid compositions of highly purified human B cell-derived exosomes. Mass spectrometric analysis indicated the abundant presence of major histocompatibility complex (MHC) class I and class II, heat shock cognate 70, heat shock protein 90, integrin ␣4, CD45, moesin, tubulin (␣ and ␤), actin, G i ␣ 2 , and a multitude of other proteins. An ␣4-integrin may direct B cell-derived exosomes to follicular dendritic cells, which were described previously as potential target cells. Clathrin, heat shock cognate 70, and heat shock protein 90 may be involved in protein sorting at multivesicular bodies. Exosomes were also enriched in cholesterol, sphingomyelin, and ganglioside GM3, lipids that are typically enriched in detergentresistant membranes. Most exosome-associated proteins, including MHC class II and tetraspanins, were insoluble in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS)-containing buffers. Multivesicular body-linked MHC class II was also resistant to CHAPS whereas plasma membrane-associated MHC class II was solubilized readily. Together, these data suggest that recruitment of membrane proteins from the limiting membranes into the internal vesicles of multivesicular bodies may involve their incorporation into tetraspanin-containing detergent-resistant membrane domains.Maturing endosomes accumulate vesicles in their lumen, resulting in their transformation into multivesicular bodies (MVB) 1 (1). These vesicles are formed by inward budding of the endosomal limiting membrane and contain a selected cargo.Proteins that are sorted to the internal vesicles of MVB potentially may have three distinct fates. The first possibility is exemplified by ligand-activated epidermal growth factor receptor, which is ultimately transferred to lysosomes for degradation (2). A second possibility is that proteins may be stored temporarily in MVB, as observed for MHC class II in immature dendritic cells (3). MHC class II-carrying MVB in dendritic cells have also been termed MHC class II compartments (MIIC), in accordance with similar structures in B cells (4). MIIC play a crucial role in peptide loading of MHC class II. In pathogen-stimulated dendritic cells, the internal vesicles of MVB fuse back with their limiting membrane, thereby allowing subsequent transfer of peptide-loaded MHC class II to the plasma membrane (3). The third potential fate of vesicles within MVB is their release into the extracellular environment as a consequence of fusion of the MVB-limiting membrane with the plasma membrane. These secreted MVB-derived vesicles have been called exosomes, which, depending on their source, may serve a multitude of functions (5-7). Exosomes are released by a great number of cell types, including reticulocytes (5), cytotoxic T cells (8), B lymphocytes (9, 10), dendritic cells (11-13), mast cells (14), platelets (15)...