Glucose homeostasis across airway epithelium maintains a glucose concentration in airway surface liquid (ASL) of ~0.4mM when blood glucose is 5mM, but rises (up to 4mM) when blood glucose is elevated up to 15mM (hyperglycaemia) making the lung more susceptible to infection1. Glucose can enter the ASL via paracellular and transcellular routes, and evidence indicates that it is removed from the ASL by glucose transporters located within the cell membrane. Intracellular glucose is maintained at low concentrations by glucose metabolism. Upon entering the cell, glucose is phosphorylated to glucose-6-phosphate (G-6-P) by hexokinase in the glycolytic pathway. G-6-P is then modified by a variety of enzymes into further metabolic products which are either utilised or expelled from the cell. This aids the maintenance of a glucose concentration gradient across the epithelial cell membrane. When intracellular glucose rises, the concentration gradient is altered which may prevent uptake of glucose from the ASL and drive glucose excretion into the ASL. Therefore, we investigated the effect of extracellular glucose on intracellular glucose concentrations to determine whether phosphorylation of glucose by hexokinase is a potential rate-determining step in maintaining ASL glucose concentrations. Data is presented as mean ± SEM and analysed by ANOVA. Using the intracellular Förster resonance energy transfer (FRET) sensor Gluconic2, a glucose binding protein flanked by donor and acceptor fluorophores cyan and yellow fluorescent proteins (CFP and YFP respectively), transfected into H441 airway epithelial cells we analysed changes in intracellular glucose by measuring absolute FRET, which increases with glucose concentration. Changing extracellular glucose concentrations from 5mM D-Glucose +10mM L-Glucose (osmotic control) to 15mM D- Glucose increased intracellular glucose as evidenced by rise in absolute FRET by 17.4 ± 2.2% (P<0.01; n=6). Addition of the GLUT transporter inhibitor Phloretin (1mM) prevented this rise in the presence of 15mM glucose. Inhibition of hexokinase II with bromopyruvic acid (100µM) after elevation to 15mM D-glucose resulted in a further increase in absolute FRET of 7.8 ± 3.7% (P<0.05; n=4) compared to 15mM glucose alone. The effect of bromopyruvic acid (100µM) when added to cells in 5mM D + 10mM L-glucose was not significant (P=0.14; n=3), These data indicate that elevating extracellular glucose concentrations results in an increase of intracellular glucose and that glucose moves into the cell via GLUT transporters. In addition, hexokinase II is important in maintaining low intracellular glucose which would promote uptake from the ASL over efflux. However, when glucose is elevated the rise in intracellular glucose could reverse this process driving more glucose into the ASL.