We investigated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) regulation by A2 adenosine (Ado) receptors and β2 adrenergic receptors in CFTR-corrected CFBE41o- airway cells and human subjects. CFBE1o- cells stimulated with Ado (10 μM), isoproterenol (Iso, 10 μM), or Ado + Iso (10 μM each) elevated cyclic AMP (cAMP) above control conditions (P < 0.001), with the Iso conditions increasing cAMP ∼ 10-fold above that produced by Ado alone (P < 0.001). All agonist conditions had similar effects on short circuit current at 10 and 25 μM, with no further currents produced by subsequent stimulation with forskolin (20 μM). CFTR dependence was demonstrated by glybenclamide block of agonist-stimulated currents. Nasal potential difference studies in normal (n = 50) subjects demonstrated that Ado (10 μM) and Ado + Iso (10 μM each) produced more polarization compared with Iso (10 μM Ado increase = 44%, 10 μM Ado + Iso increase = 52%, P < 0.05 for each condition compared with Iso alone). Studies completed in patients with CF (n = 10, "severe" genotypes) confirmed that Adostimulated polarization was CFTR-dependent. Together, these results indicate that Ado is a potent Cl- secretagogue in vivo, with relatively small effects on cAMP levels despite strong effects on CFTR-dependent short circuit current and nasal Cl- transport. These findings support growing evidence indicating a role for Ado regulation of CFTR-dependent Cl- secretion in vivo.