Dynamic modification of endosomal cargo proteins, such as the epidermal growth factor receptor, by ubiquitin can regulate their sorting into the lumen of multivesicular bodies through interactions with a complex protein network incorporating the endosomal sorting complexes required for transport (ESCRTs). Two deubiquitinating enzymes, AMSH and UBPY, interact with ESCRT protein components but exert opposite effects upon the rate of epidermal growth factor receptor downregulation. This might reflect their distinct specificities for different types of polyubiquitin chain linkage. We propose that AMSH might rescue ubiquitinated cargo from lysosomal degradation through disassembly of K63-linked polyubiquitin chains. UBPY function is essential for effective downregulation but is likely to be multifaceted, encompassing activity against both K63-linked and K48-linked polyubiquitin chains and including regulation of the stability of ESCRT-associated proteins such as STAM, by reversing their ubiquitination.