In healthy lung epithelium the main role of CFTR is Cl- secretion in airway lumen, water follows it subsequently. We recently reported that CFTR participates also in regulating of paracellular permeability of bronchial epithelial cell lines (16HBE14o-, CFBE41o-). The aim of this study was to clarify whether CFBE41o- cells overexpressing wild-type (wt CFTR) or mutant (DF508-CFTR) channels are the proper model to study paracellular transport through the epithelium. Transepithelial electrical resistance (TER) and paracellular flux of the marker molecule fluorescein were measured under resting and CFTR stimulating conditions. Stimulating of wt CFTR by increasing intracellular cyclic AMP leads to a decrease in TER of 16HBE14o- and CFBE41o- + wt CFTR cells. In contrast, in stimulated CFBE41o- and CFBE41o- + DF508 CFTR cells TER increased. Under control conditions all cell lines had similar rates of paracellular flux. Stimulation increased paracellular permeability of 16HBE14o- cell monolayers. No changes in fluorescein flux were observed in stimulated CFBE41o-, CFBE41o + wt CFTR and CFBE41o- + DF508 CFTR cells. Immunofluorescence microscopy and further morphometric analysis of the cell-cell contact length showed that 16HBE14o-cells are significantly smaller than CFBE41o- and its overexpressing clones. In consequence they have more junction contacts per area through which electrical current and solutes can leak. This influences paracellular permeability, which is controlled by tight junctions (TJ), that acts as a permeability barrier. Our results indicate that also TJ protein composition plays a role in regulating of paracellular permeability of healthy and CF cells. By overexpressing CFTR in CFBE41o- cell line it is possible to modify transcellular conductance, but the paracellular conductance is not changed. Although valuable information can be gained using CFBE41o- cells overexpressing wt CFTR and DF508 CFTR, they do not fully recapitulate the CFTR impact on paracellular pathway.