Hypercapnia is a symptom of chronic lung disease and is caused by inefficient gas exchange at the alveoli. However, the effects of elevated CO2 on airway epithelial function are not currently well understood. The airway surface liquid (ASL) plays a major role in lung innate immunity and it’s volume and composition is tightly regulated, predominantly by serous cells of the submucosal glands (SMGs) in which elevations in intracellular cAMP activate CFTR-mediated fluid and HCO3- secretion. We have previously shown that acute hypercapnia (20 minutes) reduces intracellular cAMP levels in Calu-3 cells, a human airway cell line derived from SMGs (1). The aim of the current work was to investigate the effects of chronic hypercapnia (24 hrs or more) on cAMP-dependent processes in human airway epithelia. Calu-3 cells were grown as polarised monolayers on permeable transwell supports and the volume, pH and mucus content of fluid secreted from these cells was measured over 24 hours. CFTR-dependent HCO3- transport was assessed by measuring intracellular pH changes in response to cAMP agonists in BCECF-AM loaded cells (2). In normocapnia (5% CO2), 5µM forskolin stimulated a 17.1 ± 2.6% increase in fluid secretion over 24 hours (p<0.01; n = 3), and raised the pH of the secreted fluid by 0.31 ± 0.01 (p<0.01; n = 3). However, in response to 10% CO2, the forskolin-induced fluid increase was 9.5 ± 0.9% (p<0.05 vs. normocapnia; n = 3), while the pH of the secreted fluid was unchanged (0.30 ± 0.01; p>0.05 vs normocapnia; n = 3). These results imply that CO2 exhibits differential effects on transporters regulating the volume and composition of the secreted fluid. In 5% CO2, CFTR-dependent HCO3- efflux was 232.7 ± 83.8mM/min (n = 4) which was unchanged in cells that had been exposed to 10% CO2 for 24 hours (371.3 ± 38.0mM HCO3-/min; n = 3; p>0.05 vs normocapnia) showing that chronic hypercapnia was not altering CFTR activity to produce its effects on forskolin-stimulated fluid secretion. Interestingly, 1mM IBMX stimulated a 16.8 ± 3.1% (p<0.01; n = 3) increase in fluid secretion over 24 hours which was unaffected by 10% CO2 (11.8 ± 2.9%; n = 3; p>0.05 vs normocapnia) suggesting CO2 has selective effects on cAMP generation. However, there was no effect of CO2 on mucus secretion, as measured by the Periodic Schiffs assay, in both non-stimulated (18.2 ± 1.0 µg/ml vs 19.2 ± 0.9 µg/ml; p>0.05; n = 3) and forskolin-stimulated (19.2 ± 0.1 µg/ml vs 24.0 ± 4.0 µg/ml p>0.05; n = 3) conditions.These findings demonstrate that chronic hypercapnia alters cAMP-mediated fluid but not HCO3- or mucus transport in human airway epithelia, through a CFTR-independent manner. This suggests that dysregulation of airway surface liquid secretion may occur in patients suffering from hypercapnia which could have consequences for the innate immune system of the airways.