We investigated the regulation of Cl secretion by adrenoceptors in polarized 16HBE14o- human bronchial epithelial cells. Treatment with the nonselective β adrenoceptor agonist isoprenaline stimulated an increase in short-circuit current (I ), which was inhibited by the β adrenoceptor blocker propranolol. Treatment with procaterol, an agonist specific for the β adrenoceptor subtype, stimulated a similar increase in I , which was inhibited by the β adrenoceptor antagonist ICI 118551. Inhibitors of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated Cl channel (CaCC), but not K channel blockers, were able to inhibit the increase in I . “Trimultaneous” recording of I and intracellular cyclic adenosine monophosphate (cAMP) and Ca levels in 16HBE14o- epithelia confirmed that the I induced by isoprenaline or procaterol involved both cAMP and Ca signaling. Our results demonstrate that β adrenoceptors regulate Cl secretion in the human airway epithelium by activating apical CFTRs and CaCCs via cAMP-dependent and intracellular Ca -dependent mechanisms, respectively.© 2020 Wiley Periodicals, Inc.