Hypercapnia is a symptom of chronic lung disease and is caused by inefficient gas exchange at the alveoli. We have recently shown that CO2 modulates cAMP/PKA signalling through changes in G-protein responsive transmembrane adenylyl cyclase (tmAC) activity (1). Since, cAMP signalling plays a vital role in regulating the volume and composition of the airway surface liquid (ASL) we have investigated whether changes in pCO2 impacts upon cAMP-regulated ion and fluid transport in polarised cultures of human submucosal gland derived airway epithelial cells (Calu-3). Using a radiolabelled cAMP assay, raising pCO2 from 5% to 10% for 20 mins reduced forskolin-stimulated cAMP levels by 36 ± 8.6% (n=6; p<0.05). Under normocapnic conditions, cAMP agonists produce a drop in intracellular pH of Calu-3 cells due to stimulation of CFTR-dependent HCO3- secretion (2). Acute exposure to 10% CO2 increased both the rate and magnitude of this intracellular acidification by the cAMP agonists adenosine or forskolin approximately 3 fold (n=6; p<0.05). This CO2 augmentation was independent of changes in either external or internal pH (n=3) and was prevented by either tmAC or PKA inhibition (n=4) suggesting that CO2 alters the activity of cAMP-dependent HCO3- transporters in Calu-3 cells. Pre-loading cells with the Ca2+ buffer, BAPTA-AM, and the phospholipase C inhibitor U77312 blocked the effect of 10% CO2, while carbachol, in normocapnia, mimicked the effect of hypercapnia, indicating that raising CO2 also changes cytosolic Ca2+ levels via the Gq receptor/phospholipase C pathway. Because inhibition of basolateral HCO3- transporters abolished the effect of CO2 whilst CFTR-dependent apical Cl-/HCO3- anion exchange (2) was unaffected, strongly suggests that a change in the activity of a basolateral HCO3- transporter underlies the effect of 10% CO2. Surprisingly, the cAMP-dependent HCO3- co-transporter, NBCe1, was unaffected by hypercapnia implying another, as yet unknown, basolateral HCO3- transporter is involved. Finally, exposing Calu-3 cells to chronic hypercapnia reduced forskolin-stimulated fluid secretion by 37 ± 6.1% (n=3; p<0.05). Together, these results indicate that raised CO2 decreases airway anion and fluid secretion by reducing tmAC-derived cAMP levels, and the activity of an unidentified basolateral HCO3- importer, through a process that involves crosstalk with Ca2+ signalling mechanisms. Since both the quantity and pH of the fluid bathing the airways determines the efficiency of the innate defence mechanisms of the lung, our results suggest that raised CO2 could have deleterious effects on lung function and contribute to the pathophysiology of chronic airway disease.