Cystic Fibrosis (CF) is a recessive genetic disease. CF causing mutations alter the function of the cystic fibrosis transmembrane regulator (CFTR), a cAMP activated ion channel that transports Cl- and HCO3-. CFTR is crucial for maintaining a hydrated environment for effective mucus secretion in the lungs. In CF disease, abnormal CFTR-mediated ion transport, dehydrates airway surface liquid (ASL) and mucus becomes thicker and stickier. Bacteria grow well in this changed environment encouraging chronic infections. In CF disease, pancreatic malfunction also leads to the development of cystic fibrosis related-diabetes (CFRD). This affects ~25% adolescents and ~50% of adults. In chronic obstructive pulmonary disease (COPD) the activity of CFTR has also been reported to decrease. In addition, many patients (up to 25%) suffer from Diabetes mellitus. The co-morbidity of diabetes and respiratory disease in both cases is associated with more respiratory infections, worsening lung disease and increasing the risk of respiratory failure. There is much interest in correcting mutant CFTR function in the airway using pharmacological and gene editing approaches. It is therefore important to determine the impact of the hyperglycaemic environment on CFTR function. We used Calu3 cells grown at air-liquid interface to investigate the effect of normoglycaemia (5.5mM glucose + 19.5mM mannitol) and hyperglycaemia (25mM glucose) on forskolin (10mM)/IBMX (10mM) -induced and CFTR172 inhibited CFTR activity via Ussing chamber measurement of short circuit current (Isc). Acute bilateral application of hyperglycaemia resulted in a reduction of CFTR172 sensitive Isc (17.5±3.7mA.cm-2) compared to cells exposed to normoglycaemia (8.7±2.0 mA.cm-2, p= 0.05, n = 5). However, there was no significant change in forskolin/IBMX induced Isc. As luminal glucose concentrations do not reach this level in vivo during hyperglycaemia, we exposed cell to basolateral hyperglycaemia only (which had been shown to decrease CFTR Isc in primary cells after 24 hours exposure). In this scenario there was no significant effect of acute hyperglycaemia on forskolin/IBMX-induced or CFTR172 – sensitive Isc (n=4). Furthermore, permeabilising the basolateral membrane with nystatin and applying a basolateral to apical Cl- gradient to measure apical Isc , revealed that there was no change in forskolin/IBMX-induced or CFTR172-sensitive apical Isc after either acute or 24 hours exposure of cells to basolateral hyperglycaemia compared to normoglycaemia. This data indicates that both apical and basolateral exposure of cell cultures to acute hyperglycaemia is necessary to inhibit CFTR mediated Cl- secretion, this may be a direct or indirect effect on CFTR. However, more physiological basolateral hyperglycaemia does not have direct effect on the activity of CFTR acutely or after 24 hours.