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Myosin Vb interacts with Rab11 family members and is the major motor protein identified in association with plasma membrane recycling systems in nonpolarized and polarized cells. Using yeast two-hybrid binary screens, we demonstrated that dominant active Rab25S21V fails to interact with myosin Vb, but does interact with Rab11-FIP2. Transfection of DsRed2-myosin Vb tail in MDCK cell lines stably transfected with wild-type or dominant active forms of Rab11a or Rab25 demonstrated that the distribution of Rab25S21V is only partially altered by expression of the myosin Vb tail. Finally, we demonstrate EGF-dependent sequestration of internalized EGF receptor by EGFP-myosin Vb in A431 cells. Expression of myosin Vb tail represents a powerful provocative test for the trafficking of cargoes through Rab11-containing plasma membrane recycling systems.