In Cystic Fibrosis (CF), the F508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of F508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. Here, we have hypothesized that the PDZ containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues F508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding F508-CFTR and NHE-RF1 were intranuclearly injected in A549 or MDCK cells and F508-CFTR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with F508-CFTR alone presented a low chloride channel activity whereas its co-expression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with F508-CFTR deleted of 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RF1 sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with F508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of F508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue F508-CFTR activity.