Ubiquitination induced down-regulation of cell surface proteins by internalization and lysosomal targeting plays a fundamental role in cell physiology and pathogenesis of diseases. The molecular basis of a single ubiquitin (Ub) as an autonomous endocytic signal, the widely accepted mechanism, however, remains elusive in higher eukaryotes. Using Ub containing reporter proteins without signalling abilities, we present evidence that only multiple Ub moieties, linked either covalently or assembled as oligomers with an intact interface for recognition by Ub-interacting motifs (UIMs), are recognized by the endocytic machinery in vivo and associate with a subset of Ub-binding clathrin adaptors in vitro. Genetic and pharmacological approaches show that internalization of plasma membrane proteins harbouring multiple Ub moieties is clathrin-dependent, but caveolin-independent. Functional assays demonstrate the cargo-dependent involvement of eps15/15R and epsin, UIM containing clathrin adaptors, in the endocytosis of model proteins, CD4 and the activated beta(2)-adrenergic receptor complex, containing polymeric or oligomeric Ub. These results provide a paradigm for the clathrin-mediated uptake of ubiquitinated membrane proteins in mammalian cells, requiring the assembly of multiple UIM-Ub interactions to overcome the low affinity binding of mono-Ub to UIM.