Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class that promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex while the other operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both ER-exit domains and basolateral addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable-peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well-characterized ER-exit di-acidic (DXE) motif and a tyrosine-based basolateral sorting signal conforming to YXXF motifs. This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface of transfected MDCK cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from cytosolic domain of the epidermal growth factor receptor containing a variant ER-exit motif (EXE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed for the first time that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based and an unconventional basolateral motif.