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 the measles virus and human herpes virus 6. Dlg4 is a scaffold protein important for neuronal signalling, and is a member of a family of proteins homologous to the Drosophila tumour suppressor Dlg. We show that an interaction between CD46 and Dlg4 has important implications for polarisation in epithelial cells. Specifically we show (i) biochemical evidence for a physical interaction between CD46 and Dlg4, (ii) that this interaction is specific for the Cyt1 domain of CD46 and not the Cyt2 domain, (iii) that both CD46 and an alternatively spliced isoform of Dlg4 are polarised in normal human epithelial cells, and (iv) that the polarised expression of CD46 in epithelial cells requires the Dlg4 binding domain and alters with the ectopic expression of a truncated mutant form of Dlg4. This is the first identification of a direct, and cytoplasmic domain-specific interaction between CD46 and an intracellular signalling molecule, and indicates a molecular mechanism for the polarisation of CD46. These data also indicate that, in addition to the known role for Dlg4 in neuronal cells, Dlg4 may be important for polarisation in epithelial cells.