A novel technique for the biotinylation of inner membrane vesicles has been developed to enable the purification of E. coli inner membrane vesicles utilizing a high affinity biotin-streptavidin interaction. Previous studies have demonstrated recovery and purification of synthetic lipid vesicles utilizing biotinylated synthetic lipids; however, this previous method is not suitable for vesicles obtained from living cells. In this work, inner membrane vesicles obtained from E. coli cells were biotinylated and then immobilized to a streptavidin resin. A binding capacity of 0.4 mg vesicle per ml of resin was observed, comparable to the binding capacity reported for synthetic vesicles. This technique is directly applicable to the recovery of membrane proteins from complex cell lysate and also allows the immobilization of membrane vesicles for a variety of applications.