The pH of the airway surface liquid (ASL) is tightly regulated to maintain optimal conditions for clearance of pathogens. In Cystic Fibrosis (CF), ASL pH is more acidic, thought to be due to reduced bicarbonate secretion1. This leads to impaired activity of antimicrobial peptides and defective bacterial killing in animal models of CF lung infections1. In addition ASL glucose concentrations are raised in CF and further in CF-related diabetes, which is associated with increased colonisation with P. aeruginosa 2,3. The aim of this study was to investigate how the airway epithelium responds to acidic metabolic by-products produced by P. aeruginosa (PA01), a key pathogen in CF lung disease, and whether this is altered in CF cultures with and without hyperglycaemia. ASL pH and bacterial growth was monitored across primary human non-CF and CF bronchial epithelial cells (HBE) and Calu-3 cell line, cultured at air-liquid-interface, over 6 hours. Calu-3 and non-CF HBE cells were able to maintain ASL pH in the presence of P. aeruginosa (n=6). However ASL pH across CF HBE cells became more acidic after P. aeruginosa addition (decreased by 0.3 pH units after 6 hours, p<0.05, n=5). This acidification was enhanced by increasing the glucose content of the bathing solution (from 5 to 15mM), which correlated with increased P. aeruginosa growth (18±4% increase, p<0.05, n=5). ASL acidification in the presence of P. aeruginosa could be replicated in non-CF HBE and Calu-3 cultures by replacing bicarbonate with a HEPES-buffered bathing solution in order to limit epithelial bicarbonate secretion. Similar to CF cultures, increasing glucose led to a greater ASL acidification across infected non-CF HBE cells in the presence of HEPES (p<0.05, n=5). Replacing bicarbonate with HEPES increased P. aeruginosa growth across all cell types and growth was further enhanced by raising glucose concentration of the bathing solution (p<0.01, n=5). These results indicate that P. aeruginosa metabolic by-products stimulate airway epithelial bicarbonate secretion in order to maintain ASL pH optimal for bacterial killing. This process is impaired in CF epithelium leading to ASL acidification and bacterial growth both of which are enhanced by elevation of ASL glucose concentration. Overall the data are consistent with a critical role for CFTR-dependent bicarbonate secretion in airway innate immunity and the suppression of pathogen growth.