 Extracellular vesicles, EVs, play a role in intercellular communication, however the precise biogenesis of different populations of EVs are not clear. Here, the authors follow the intracellular trafficking of two proteins before their secretion in EVs and report the biogenesis and protein markers of EV subtypes, ectosomes budding from the plasma membrane as well as exosomes from late endosomes. This article was authored by Matilde Machu, Natalie Nevo, Mabel Duve, and others.