Research on epithelial cell lines and primary epithelium is required to dissect the mechanisms underlying the structural abnormalities in airway epithelium observed for respiratory diseases, including asthma and chronic obstructive pulmonary disease. The novel electric cell-substrate impedance sensing technique was used to monitor cell adhesion/spreading, barrier function and wound healing. Primary bronchial epithelium was compared with airway epithelial cell lines 16HBE14o-, BEAS-2B, NCI-H292 and A549. BEAS-2B, A549 and primary cells form a confluent monolayer more rapidly than do 16HBE14o- cells. In contrast, 16HBE14o- cells form stronger intercellular contacts, with a 10-fold higher resistance than BEAS-2B, A549 and NCI-H292 cells and a five-fold increase over primary cells. Accordingly, expression of the adhesion molecules zona occludens-1 and E-cadherin was highest in 16HBE14o- cells. These molecules were localised in intercellular junctions in both 16HBE14o- and primary cells. Finally, restoration of barrier function upon injury was impaired in BEAS-2B compared to 16HBE14o- cells. In conclusion, epithelial cell types display remarkable phenotypic differences and should, accordingly, be used to address specific research questions. 16HBE14o- cells appear most suitable for studies on barrier formation, whereas resemblance in attachment of primary and BEAS-2B and A549 cells makes the latter more important for translational research on cell-matrix contact.