Introduction and Objectives: Cystic fibrosis (CF) results from alterations in the CF transmembrane conductance regulator (CFTR) gene but the exact pathogenesis of lung disease remains poorly understood. Ceramide is an essential constituent of plasma membranes and regulates many cellular responses. It has recently been shown that CFTR-deficient mice accumulate ceramide in airway epithelial cells resulting in inflammation and susceptibility to Pseudomonas aeruginosa – two key features of CF lung disease.¹ Ceramide accumulation was also demonstrated in nasal epithelial cells from people with CF and qualitatively in a small number of lower airway sections.¹ The objective of this work was to evaluate ceramide levels quantitatively in the lower airway epithelium of people with CF compared to pulmonary hypertension (PH), emphysema and unused donors and to investigate relationships between epithelial ceramide and markers of neutrophilic inflammation and P. aeruginosa infection. Methods: Immunohistochemistry was performed on airways from explanted lungs (8 CF, emphysema and PH respectively) and 8 donor lungs using ceramide (Glycobiotech), neutrophil elastase (NE) and myeloperoxidase (MPO) antibodies. Ceramide staining was evaluated in the lower airway epithelium in a blinded fashion using image analysis software and expressed as percentage area. A mean from 5 randomly selected, non-overlapping high-power fields was used for each patient. The number of cells staining positive in the airway for NE and MPO/millimetre basement membrane was also evaluated. Differences were compared using the Mann-Whitney test. Results: Staining for ceramide was significantly increased in the lower airway epithelium of people with CF (median 14.11%, range 8.32-16.2) compared to PH (3.03%, p=0.0009, 2.05-5.72), unused lung donors (3.44%, p=0.0009, 1.86-4.9) and emphysema (5.06%, p=0.01, 1.52-13.24) (Figure 1). Ceramide staining was increased in emphysematous lungs compared to PH (p=0.0135) and unused donors (p=0.0009). The number of NE and MPO positive cells in the airway was positively correlated with the percentage of epithelium staining for ceramide (Pearson correlations 0.634, p<0.000 and 0.704, p<0.000 respectively). Ceramide staining was significantly increased in lungs colonised with P. aeruginosa (median 10.1%, range 1.52-16.2) compared to those not colonised (3.14% p=0.0106, 2.05-10.6). Conclusions: Ceramide accumulates in the lower airway epithelium of people with CF, and to a lesser extent in emphysema, and is positively correlated with markers of neutrophilic inflammation and P. aeruginosa infection. This data supports the hypothesis that ceramide plays a role in the pathogenesis of CF lung disease and may represent a target for pharmacotherapy.