The study of clathrin-coated vesicles (CCVs) isolated from various organs has revealed the identities and important features of many of the factors involved in membrane trafficking. The development of isolation methods using cultured cell lines has made it possible to manipulate the source material before isolation to ask important questions about the roles of these factors and the pathways in which they are involved. We discuss here the advantages and limitations of the use of cultured cell lines for the isolation of CCVs.