One of the main goals of this study was to understand the relationship between an epidermal growth factor (EGF) receptor dileucine (LL)-motif (679-LL) required for lysosomal sorting and the protein ubiquitin ligase CBL. We show that receptors containing 679-AA (di-alanine) substitutions that are defective for ligand-induced degradation nevertheless bind CBL and undergo reversible protein ubiquitylation similar to wild-type receptors. We also demonstrate that 679-LL but not CBL is required for EGF receptor downregulation by an endosomal membrane protein encoded by human adenoviruses that uncouples internalization from post-endocytic sorting to lysosomes. 679-LL is necessary for endosomal retention as well as degradation by the adenovirus protein, and is also transferable to reporter molecules. Using NMR spectroscopy, we show that peptides with wild-type 679-LL or mutant 679-AA sequences both exhibit alpha-helical structural propensities but that this structure is not stable in water. A similar analysis carried out in hydrophobic media showed that the alpha-helical structure of the wild-type peptide is stabilized by specific interactions mediated by side-chains in both leucine residues. This structure distinguishes 679-LL from other dileucine-based sorting-signals with obligatory amino-terminal acidic residues that are recognized in the form of an extended beta or beta-like conformation. Taken together, these data show that 679-LL is an alpha-helical stabilizing motif that regulates a predominant step during lysosomal sorting, involving intracellular retention under both sub-saturating and saturating conditions.