Multiple sorting steps within eukaryotic cells are mediated by tyrosine-based sorting motifs. Motifs conforming to the consensus -YXXO- (where O indicates a bulky hydrophobic residue) have been shown to specify high-efficiency internalization from the plasma membrane, targeting from the plasma membrane to the trans-Golgi network and targeting to lysosomal compartments as well as being involved in basolateral sorting in polarized cells. These motifs are recognized by the medium-chain subunits of heterotetrameric adaptor complexes. Whereas these motifs have been shown to be sufficient to mediate interaction with the mu-chains, we and others have shown that their context is important in determining the affinity of interaction. In this study we have investigated the interaction between the tyrosine motifs of the type-1 integral membrane proteins TGN38 and lgp120 with medium-chain subunits using the yeast two-hybrid system. Whereas the wild-type version of the cytosolic domain of TGN38 interacts with highest affinity with mu2, we show that the cytosolic domain of lgp120 interacts almost exclusively with mu3A. The specificity of binding of tyrosine-based sorting motifs to mu-chains is shown to be highly sensitive to the context in which the motif lies. For example, the -YQTI- motif of lgp120 is effectively non-functional with regard to mu-chain binding when placed in the context of the TGN38 cytosolic domain. Deletion of four amino acids (NLKL) at the extreme C-terminus of TGN38, leaving the YXXO motif as the C-terminus, greatly enhances the affinity of interaction with mu2. Furthermore, addition of these same residues to the extreme C-terminus of lgp120 effectively abolishes the interaction of the cytosolic domain of lgp120 with mu-chains. We also show that the newly identified mu-adaptin-related protein 2 (mu4) only interacts weakly with tyrosine-based sorting motifs.