A microscopic layer of secreted fluid covers the airways and provides the first line of defense against inhaled pathogens. Depletion of this airway surface liquid (ASL) in cystic fibrosis (CF) disrupts mucociliary clearance and leads to chronic infections by Pseudomonas aeruginosa and other bacteria. Epithelial bicarbonate secretion is also reduced in CF, resulting in a decrease in pH that may promote infection in CF pig airways, although it remains uncertain whether the measured reduction from pH 7.14 to 6.96 inhibits antimicrobial activity significantly. We found that Pseudomonas is sensitive to alkaline pH in pure solutions when pH is elevated by a decrease in luminal pCO2 or increase in HCO3- concentration. Computer simulations suggested that CO2, which varies between 0.035% during inspiration and 5% during expiration, may equilibrate with ASL. To test this experimentally we studied thin layers of liquid placed on monolayers of fixed Calu-3 cells, a human airway epithelial cell line Calu-3. BCECF-dextran microfluorimetry was used to measure surface liquid pH, a computer-controlled gas mixing pump was used to generate pCO2 oscillations that simulate tidal breathing, and pCO2 was monitored using a fast response CO2 analyzer. Physiological pCO2 oscillations caused the surface pH to vary by ~1.5 units, with pH reaching maximal values of pH 8.7 – 9.2 depending on HCO3- concentration. Similar values were observed under steady-state conditions when secretions were equilibrated with 5% CO2 (7.42± 0.11, mean ± s.d., n=8) or room air (9.2 ± 0.14; n=7). The antimicrobial effect of oscillating pH was assessed by comparing by counting colony forming units on agar plates after exposure to physiological CO2 oscillations or various (constant) pCO2 levels for 1, 3, or 6 hours at 37oC. The number of viable bacteria increased 3-fold after 6h exposure to 5% CO2 but not 2.5% CO2, presumably due to moderate elevation of the pH. Oscillations between 5% and 0.035% were also bacteriostatic when secretions were collected from unstimulated Calu-3 cells. Importantly, 90% killing was observed if fluid was collected from forskolin-stimulated control Calu-3 cells, but not if fluid was collected from CFTR knock-down cells. Similar results were obtained using other HCO3- secretagogues (VIP, cpt-cAMP) and with mucoid and non-mucoid strains of Pseudomonas. We conclude that HCO3- allows rapid, transient alkalinizations during physiological variations in pCO2. Bacteria are sensitive to alkaline pH, and this sensitivity is enhanced by pCO2 oscillations and by stimulated HCO3- secretion. These results provide the first evidence for dynamic alterations in airway surface pH and help clarify the role of CFTR and bicarbonate secretion in airways host defense.