In healthy bronchial epithelium the activity of CFTR establishes a salt gradient and water follows osmotically through transcellular and paracellular pathways. In cystic fibrosis (CF) salt and water transport in bronchial epithelium is impaired, which results in mucus adhesion and chronic bacterial colonization. The aim of the study was to clarify if and how CFTR participates in regulating of paracellular permeability. Transepithelial electrical resistance (TER) and paracellular flux of the marker molecule fluorescein were measured under resting and CFTR stimulating conditions. We could show by measurements under short circuit conditions, that transepithelial potential difference does not influence fluorescein flux. Stimulation of wt-CFTR-expressing cells (16HBE14o-) by cAMP elevating agents produced a dramatic decrease of TER. This effect was paralleled by an increase in the paracellular permeability. By contrast, the paracellular permeability of DF508-CFTR-expressing cells (CFBE41o-) decreased and TER increased transiently upon cAMP elevation. This suggests that cAMP reduced paracellular permeability, as shown by an increase of TER in CFBE41o- cells. In wt-CFTR expressing cells this reduction of paracellular permeability was not only masked by a strong increase of transcellular ion permeability, but overwhelmed by a paracellular effect of CFTR activity. We assume that ATP, released by CFTR, regulates paracellular permeability by an autocrine purinergic pathway. This hypothesis was supported by the finding, that the application of ATP/UTP produced an increase of paracellular permeability of both 16HBE14o- and CFBE41o- cells. In summary, our data suggest that CFTR plays a crucial role in regulating airway bronchial epithelial paracellular permeability.