Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl- channels. Thus, CF epithelia fail to transport Cl- and water. A postulated therapeutic avenue in CF is activation of alternative Ca2+-dependent Cl - channels. We hypothesized that stimulation of Ca2+ entry from the extracellular space could trigger a sustained Ca2+ signal to activate Ca2+-dependent Cl- channels. Cytosolic [Ca2+]i was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o-) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (ΔF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca2+ in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca2+ entry stimulated sustained Cl- and HCO3- secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl- secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl- permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.