Recent work suggests activation of the Ca2+ activated chloride ion channel TMEM16A (anoctamin 1) as a genotype-independent (mutation-agnostic) treatment for cystic fibrosis, and also for other inflammatory airway diseases. This strategy is based on data obtained in studies performed almost exclusively in vitro, or under non-inflammatory conditions in vivo. It is assumed that activation of TMEM16A will induce airway electrolyte secretion, thereby compensating for the defect in CFTR-mediated Cl- secretion. This should then lead to reduced airway mucus plugging and improved mucociliary clearance. However, in healthy lungs of human and mouse, TMEM16A is only weakly expressed, while in airways of patients with asthma and cystic fibrosis (CF), and in lungs of asthmatic mice, TMEM16A is upregulated particularly in glands and airway smooth muscle cells. Activation/potentiation of TMEM16A in these cells may enhance mucus plugging and induce bronchoconstriction, which would favor a therapeutic use of inhibitors of TMEM16A, rather than activators. We analyzed expression of TMEM16A at different locations in human and mouse airways. We find upregulation of TMEM16A in lungs of people with CF or in asthmatic lungs, particularly in submucosal glands. However, in CF submucosal cells, stimulation by the purinergic agonist ATP activated predominantly KCNN4 K+ channels, but not ANO1. Niclosamide, an inhibitor of TMEM16A, blocked mucus production and mucus secretion in mice in vivo and in vitro. In contrast, the activator/potentiator of TMEM16A, Eact, and the potentiator of TMEM16A, brevenal, both induced acute mucus release from airway goblet cells in mice in vivo. Brevenal, a bioactive compound from the marine dinoflagellate Karenia brevis, strongly potentiated ATP-activated Cl- currents, without directly increasing cytosolic Ca2+. Both Eact and brevenal induced acute airway contraction. Treatment of airway epithelial cells with niclosamide strongly inhibited expression of the central transcription factor for Th2 inflammation and goblet cell differentiation, SAM-pointed domain–containing ETS-like factor (SPDEF). Taken together, activators/potentiators of TMEM16A may induce preferentially airway mucus secretion and bronchoconstriction, which suggests the use of TMEM16A-inhibitors for the treatment of CF lung disease. All animal experiments were approved by the local Ethics Committee and were conducted according to the guidelines of the American Physiologic Society and the German Law for the Welfare of Animals.