Rab GTPases are key regulators of vesicular protein transport in both the endocytic and exocytic pathways. In endocytosis and recycling, Rab11 plays a role in receptor recycling to plasma membrane via the pericentriolar recycling compartment. However, little is known about the molecular requirements and partners that promote transport through Rab11-positive recycling endosomes. Here, we report a novel approach to reconstitute transport to immunoabsorbed recycling endosomes in vitro. We show that transport is temperature-, energy-, and time-dependent and requires the presence of Rab proteins, as it is inhibited by the Rab-interacting protein Rab GDP-dissociation inhibitor that removes Rab proteins from the membrane. Cytochalasin D, a drug that blocks actin polymerization, inhibits the in vitro assay, suggesting that transport to recycling endosomes depends on an intact actin cytoskeleton. Using an affinity chromatography approach we show the identification of Rab11-interacting proteins including actin that stimulate transport to recycling endosomes in vitro. (C) 2003 Elsevier Inc. All rights reserved.