Submucosal glands are the major mucus-secreting cells in the tracheobronchial tree, and they appear to be affected in cystic fibrosis (CF). To study the dysregulation of pulmonary secretion in CF, human tracheal glandular (HTG) cells were isolated from tracheal mucosa of CF patients undergoing bipulmonary transplantation and compared with normal HTG cells. The cells were cultured in Dulbecco's modified Eagle's-Ham's F-12 medium supplemented with Ultroser G, on collagen type 1-coated dishes. We observed that the secretion rates for the three specific serous secretory markers: bronchial inhibitor (BrI), lysozyme, and lactoferrin were 10, 20, and 50 times higher, respectively, in CF-HTG cells than in normal HTG cells. Furthermore, the two physiological neurotransmitters: acetylcholine and norepinephrine, which have opposite actions on the secretion of BrI (suggesting that these neurotransmitters acted as regulators of secretion) did not induce the significant modification of protein secretion observed with normal HTG cells. In combination with forskolin and calcium ionophore A23187, secretion of BrI was minimally modified, indicating a lack of responsiveness of CF-HTG cells to these agonists. In conclusion, CF-HTG cells in culture show a constitutive hypersecretion and an hyporesponsiveness to agonists. They provide a useful tool to study the regulation defect of bronchial secretion observed in CF.