Extracellular vesicles (EVs) are small lipid vesicles secreted by most cell types. They originate either from the cell surface (and typically labelled with tetraspanin CD9) or from multivesicular bodies that release EVs by fusion with the cell surface (and typically labelled with tetraspanin CD63). Isolated and concentrated EVs can be taken up by acceptor cells. However, this approach do not allow neither the analysis of the distance to which EVs can be transferred, nor the number of EVs that can be transferred from one cell to another. Here we used MCF7 cells as a model of mammary epithelial cells in which some of the mechanisms of biogenesis of EVs has been described3. By adapting a coculture system we developed previously, we studied the amount, the localisation and distance of the transfer of EV classical markers by 3D high resolution confocal microscopy. Our coculture approach showed the release of the three tetraspanins studied here resulted in their adhesion to the substratum and transfer to only neighbouring cells. This approach will be useful to further decipher the mechanism underlying the transfer and uncovering previously unrecognized EV cargoes.