Due to the cellular complexity of the airway epithelium, it is important to carefully define bronchial cell lines that capture the phenotypic traits of a particular cell type. We describe the characterization of a human bronchial epithelial cell line, VA10. It was established by transfection of primary bronchial epithelial cells with retroviral constructs containing the E6 and E7 oncogenes from HPV16. The cell line has been cultured for over 2 yr, a total of 60 passages. Although prolonged culture resulted in increased chromosomal instability, no major phenotypic drift in marker expression was observed. The cells expressed cytokeratins 5, 13, 14, and 17 suggesting a basal-like phenotype. This was further supported by the expression of α6β4 integrins and the basal cell-associated transcription factor p63. The VA10 cell line generated high trans-epithelial electrical resistance in suspended and air–liquid interface culture, indicating functionally active tight junction (TJ) complexes. Immunocytochemistry showed the typical reticular structures of occludin and TJ-associated F-actin. VA10 produced pseudostratified layer in air–liquid interface culture with expression of p63 restricted to the basal layer. Furthermore, VA10 produced round colonies when cultured in laminin-rich reconstituted basement membrane, and immunostaining of claudin-1 and the baso-lateral marker β4 integrin revealed colonies that generated polarization as expected in vivo. These data indicate that VA10 epithelia have the potential to model the bronchial epithelium in vivo and may be useful to study epithelial regeneration and repair and the effect of chemicals and potential drug candidates on TJ molecules in airway epithelia.