The objective of this study was to examine the influence of cystic fibrosis transmembrane conductance regulator (CFTR) on paracellular permeability of human lung epithelium. The widely used cell lines Calu-3 (submucosal glands), wtCFTR expressing 16HBE14o^- and DF508-CFTR expressing CFBE41o^- bronchial epithelium cells where grown to confluence on permeable supports. Transepithelial resistance (TER) and paracellular flux of the marker molecules fluorescein and ¹⁴C-Mannitol were measured in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Under resting conditions, the paracellular permeability of Calu-3 cells and 16HBE14o^- are comparable and about two times higher compared to CFBE41o^- monolayers. Stimulation of wtCFTR expressing cell lines by cAMP produced a dramatic decrease of transepithelial electrical resistance (TER) of about 50% within 5 min. This effect was concomitant with a decrease in the paracellular permeability of the marker molecule fluorescein and ¹⁴C-Mannitol by 40%. Contrarily the TER of DF508-CFTR expressing CFBE41o^- cells increases by 10% while their paracellular permeability shows no statistically significant change. From these data we conclude that in wtCFTR expressing lung epithelium paracellular permeability is high under basal conditions allowing passive osmotic adjustment of the airway surface liquid (ASL) by salt and water movement through the paracellular pathway. Under CFTR stimulating conditions (cAMP) lung epithelium secretes actively salt and water into apical side. Simultaneously the paracellular permeability is reduced to avoid reflux of salt and water to the basal side. In CF epithelia the paracellular permeability is low under basal and stimulating conditions due to the absence of CFTR. As a result no passive osmotic adjustment of the ASL can occur. In summary, our data show that airway epithelium lacking functional CFTR expression is unable to control the paracellular transport.