The cystic fibrosis transmembrane conductance regulator (CFTR) is an integral membrane glycoprotein which functions as an anion channel and influences diverse cellular processes. We studied its role in the development of epithelial tightness by expressing wild-type (WT-CFTR) or mutant (Delta F508-CFTR) CFTR in human airway epithelial cell monolayers cultured at the air-liquid interface. Green fluorescent protein (GFP)-tagged WT or Delta F508 constructs were expressed in the CF bronchial cell line CFBE41o(-) using adenoviruses, and the results were compared with those obtained using CFBE41o(-) lines stably complemented with wild-type or mutant CFTR. As predicted, GFP-Delta WT-CFTR reached the apical membrane whereas GFP-F508-CFTR was only detected intracellularly. Although CFTR expression would be expected to reduce transepithelial resistance (TER), expressing GFP-CFTR significantly increased the TER of CFBE41o(-) monolayers whilst GFP-Delta F508-CFTR had no effect. Similar results were obtained with cell lines stably overexpressing Delta F508-CFTR or WT-CFTR. Preincubating Delta F508-CFTR monolayers at 29 degrees C reduced mannitol permeability and restored TER, and the effect on TER was reversible during temperature oscillations. Expression of GFP-Delta F508-CFTR or GFP-WT-CFTR in a cell line already containing endogenous WT-CFTR (Calu-3) did not alter TER. The CFTR- and temperature-dependence of TER were not affected by the CFTR inhibitor CFTR(inh)172 or low-chloride medium; therefore the effect of CFTR on barrier function was unrelated to its ion channel activity. Modulation of TER was blunted but not eliminated by genistein, implying the involvement of tyrosine phosphorylation and other mechanisms. Modulation of CFTR trafficking was correlated with an increase in tight junction depth. The results suggest that CFTR trafficking is required for the normal organisation and function of tight junctions. A reduction in barrier function caused by endoplasmic reticulum retention of Delta F508-CFTR may contribute to fluid hyperabsorption in CF airways.