We have developed an in vitro assay to reconstitute the formation of endosomal recycling vesicles. To achieve specificity for endosomes as the donor organelle, cells are surface-biotinylated and allowed to endocytose for 10 min, after which the remaining surface-biotin is stripped off. The cells are then permeabilized and the cytosol washed away. Upon addition of exogenous cytosol and energy, sealed vesicles containing biotinylated recycling receptors are produced. Modification of the cytosol, for example, by immunodepletion or addition of purified proteins, allows the identification of proteins involved in vesicle formation. The results show that recycling is mediated by AP-1/clathrin-coated vesicles, requires Rab4, and is negatively regulated by rabaptin-5/rabex-5.