The cystic fibrosis transmembrane conductance regulator (CFTR) operates in a macromolecular complex at the apical membranes of epithelial cells. Complex formation is facilitated by interaction with the PDZ scaffolding protein EBP50 via a C-terminal PDZ binding motif in CFTR. The C-terminal motif participates in endocytic recycling and apical localization but there is evidence that other trafficking signals exist in CFTR (Ostedgaard et al., 2003;Milewski et al., 2001;Swiatecka-Urban et al., 2002). Using GST pulldowns, mass spectrometry and protein overlay assays, we have identified an internal six amino acid region (EK) in CFTR that binds EBP50 and, in conjunction with the C-terminal PDZ binding motif, enhances EBP50 multimerization. We have characterized the cellular responsibility of this EK motif by ascertaining the steady state subcellular location of both transiently and stably expressing CFTR-EK mutants in polarized cell lines via membrane biotinylation and confocal microscopy. Mutation of the EK motif results in aberrant trafficking, with CFTR accumulating at the Golgi complex and the endocytic recycling compartment. Together, these results indicate that CFTR directed multimerization of EBP50 is integral to endocytic recycling and localization of CFTR to apical membranes.