In human airways, the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is predominantly expressed in serous cells of the tracheobronchial glands. Despite considerable evidence that submucosal glands are important contributors to the pathophysiology of CF lung disease, most attempts at CFTR gene transfer have primarily targeted airway surface epithelial cells. In this study, we systematically evaluated CFTR gene transfer into cultures of immortalized CF human tracheobronchial submucosal gland (6CFSMEO) cells using adenovirus and cationic lipid vectors. We found that the efficiency of adenovirus-mediated gene transfer was comparable in 6CFSMEO and CFT1 cells (a surface airway epithelial cell line isolated from a subject with CF). So was the ranking order of adenovirus vectors containing different enhancers/promoters (CMV >> E1a approximately phosphoglycerokinase), as determined by both X-Gal staining and quantitative measurement of beta-galactosidase activity. Further, we provide the first demonstration that cationic lipids mediate efficient gene transfer into 6CFSMEO cells in vitro. The transfection efficiency at optimal conditions was higher in 6CFSMEO than in CFT1 cells. Finally, either infection with adenoviral vectors or transfection with cationic lipid:plasmid DNA complexes encoding CFTR significantly increased chloride (Cl-) permeability, as assessed using the 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) fluorescence assay, indicating restoration of functional CFTR Cl- channel activity. These data show that although the mechanisms of transfection may be different between the two cell types, 6CFSMEO cells are as susceptible as CFT1 cells to transfection by adenoviral and cationic-lipid gene transfer vectors.