Using a yeast two-hybrid screen, we identified a physical interaction between CD46 and DLG4. CD46 is a ubiquitous human cell-surface receptor for the complement components C3b and C4b and for measles virus and human herpesvirus 6. DLG4 is a scaffold protein important for neuronal signaling and is homologous to the Drosophila tumor suppressor DLG. We show that an interaction between CD46 and DLG4 is important for polarization in epithelial cells. Specifically, we show (i) biochemical evidence for an interaction between CD46 and DLG4, (ii) that this interaction is specific for the Cyt1 (but not Cyt2) domain of CD46, (iii) that both CD46 and an alternatively spliced isoform of DLG4 are polarized in normal human epithelial cells, and (iv) that the polarized expression of CD46 in epithelial cells requires the DLG4-binding domain and alters with expression of a truncated form of DLG4. This is the first identification of a direct and cytoplasmic domain-specific interaction between CD46 and an intracellular signaling molecule and provides a molecular mechanism for the polarization of CD46. These data also indicate that, in addition to the known role for DLG4 in neuronal cells, DLG4 may be important for polarization in epithelial cells.