Chloride secretion by airway epithelial cells is defective in cystic fibrosis (CF). The conventional paradigm is that CFTR is activated through cAMP and protein kinase A (PKA), whereas the Ca2+-activated chloride channel (CaCC) is activated by Ca2+agonists like UTP. We found that most chloride current elicited by Ca2+agonists in primary cultures of human bronchial epithelial cells is mediated by CFTR by a mechanism involving Ca2+activation of adenylyl cyclase I (AC1) and cAMP/PKA signaling. Use of selective inhibitors showed that Ca2+agonists produced more chloride secretion from CFTR than from CaCC. CFTR-dependent chloride secretion was reduced by PKA inhibition and was absent in CF cell cultures. Ca2+agonists produced cAMP elevation, which was blocked by adenylyl cyclase inhibition. AC1, a Ca2+/calmodulin-stimulated adenylyl cyclase, colocalized with CFTR in the cell apical membrane. RNAi knockdown of AC1 selectively reduced UTP-induced cAMP elevation and chloride secretion. These results, together with correlations between cAMP and chloride current, suggest that compartmentalized AC1–CFTR association is responsible for Ca2+/cAMP cross-talk. We further conclude that CFTR is the principal chloride secretory pathway in non-CF airways for both cAMP and Ca2+agonists, providing a novel mechanism to link CFTR dysfunction to CF lung disease.