Cystic Fibrosis (CF) is the most frequent genetic disease in the Caucasian population. CF is caused by a defective gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). The CFTR influences the fluid composition of the mucus in the respiratory tract and the most common mutation inducing CF is DF508. This mutation impairs the CFTR processing within the cell and also prevents functional CFTR expression in the apical membrane. The aim of this study is to investigate the functional expression and insertion of wild type (wt) CFTR in the apical plasma membrane of human CF airway epithelia after wt-CFTR-mRNA transfection. The human bronchial epithelial cell line CFBE41o- stably expressing DF508-CFTR is well suited for CF research: It forms polarized cell monolayers with a sufficient transepithelial resistance when grown on permeable filters that allows functional measurements of transepithelial transport processes. The cells can be mounted in modified Ussing chambers for electrophysiological measurements to determine transepithelial short-circuit current, conductance and capacitance. In orientating experiments we determined the appropriate length of the CFTR-mRNA PolyA-tail for an effective and long lasting expression by using the Xenopus laevis oocyte expression system. In two electrode voltage clamp experiments we showed that a 120 PolyA-tail produces maximal CFTR current and conductance and the capacitance increases following cAMP stimulation. Further on, we carried out the Ussing chamber experiments with CFBE41o- cells transfected with this CFTR-mRNA. In contrast to the non-transfected cells, the transfected cells showed an increased CFTR current, conductance and capacitance after CFTR activation by cAMP (100 mM). Thus, CFTR currents in CFTR-mRNA transfected cells were identical with cells natively expressing CFTR. The increase in capacitance shows that a major part of this activation is caused by exocytotic insertion of preformed functional CFTR molecules into the apical membrane. The CFTR activation is sensitive to the Cl- channel blocker NPPB, which decreased all parameters significantly. Furthermore, we determined the abundance of whole cell CFTR protein in transfected CFBE41o- cells by Western blot analyses and additionally, we detected CFTR molecules just at the apical surface of these cells with immunofluorescence approaches. From these data we conclude that CFTR-mRNA transfection could be a novel tool in gene therapy to restore impaired CFTR function.