PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of CFTR, an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C-terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNAi targeting of endogenous CAL specifically increases cell-surface expression of the disease-associated F508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL:CFTR interaction from purified components in vitro, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL:CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturation trafficking defects in cystic fibrosis.