The airway epithelium is covered by a thin layer of surface liquid (ASL), which is vital for maintaining the sterility of the respiratory tract. The glucose concentration of the ASL (~0.4mM) is much lower than that in blood and is tightly regulated by the airway epithelium (1). ASL glucose increases with hyperglycaemia and respiratory disease (including cystic fibrosis and chronic obstructive pulmonary disease) and this increases the risk of respiratory bacterial infection (2,3). ASL lactate concentrations also rise with hyperglycaemia and respiratory inflammation. The aim of this study was to investigate whether the airway epithelium secretes lactate into ASL under hyperglycaemic conditions and if this contributes to changes in ASL pH due to lactate-H+ co-transport.H441, Calu-3 and primary human bronchial epithelial cells were grown on permeable supports at air-liquid-interface to form confluent polarised monolayers. Hyperglycaemia was mimicked by increasing the glucose concentration of the HCO3–buffered physiological salt solution in the basolateral chamber from 5 to 15 mM. ASL lactate was sampled after 6 hours by washing the apical surface with 50 µl of salt solution and measured using a lactate assay kit (Sigma, UK). ASL pH was measured by adding 100 µl of salt solution to the apical surface and using a pH microelectrode. Increasing basolateral glucose resulted in a significant rise in ASL lactate across all airway epithelial cell types (P < 0.05, n=5-12), but no significant changes in basolateral lactate concentrations were observed. ASL pH of H441 monolayers decreased by 0.09 ± 0.02 pH units (P < 0.05, n=4), consistent with lactate-H+ co-transport acidifying the ASL. However, an alkalinisation of 0.10 ± 0.05 pH units (P < 0.05, n=9) was observed in the ASL of hyperglycaemic Calu-3 monolayers. As Calu-3 cells produce HCO3–rich fluid secretions, ASL acidification could stimulate HCO3- secretion to neutralise the ASL. To test this hypothesis, the experiment was repeated using a HEPES-buffered salt solution to reduce epithelial HCO3- secretion. Under these conditions, Calu-3 ASL pH decreased in response to elevated glucose. Inhibition of monocarboxylate transporters MCT1 and MCT2 (lactate-H+ co-transport) with AR-C155858 (100 nM) partially overcame the effects of hyperglycaemia on ASL lactate and pH, although the results indicate that other transporters may be involved.These data indicate that hyperglycaemia increases airway epithelial lactate secretion, in part via MCT1/2 lactate-H+ co-transport, which can acidify the ASL in the absence of epithelial HCO3- secretion. This could be important in the pathogenesis of cystic fibrosis as both airway glucose and lactate concentrations are elevated and ASL pH is more acidic due to aberrant HCO3- secretion.