Maintaining the integrity of the airway epithelial barrier is essential in preventing increased permeability to harmful molecules and pathogens, which may lead to increased risk of developing respiratory infection and disease. We have previously shown that in human airway epithelial cells metformin increases tight junction (TJ) assembly after pathogen disruption via an AMPK-PKCζ pathway1-2. The sphingomyelinase/ceramide system can cleave membrane proteins releasing ceramide to form ceramide rich domains, signaling the reorganization of receptors and activation of protein kinase C isoforms such as PKCζ3. This study aimed to investigate the effect of N-Palmitoyl-d-erythro-sphingosine (C16:0 ceramide), a PKCζ activator on epithelial integrity of human airway epithelial cells (H441 cells). We investigated the effect of ceramide on claudin-3 which can alter ion selectivity4. H441 cells were grown on permeable supports at air-liquid interface, TJ were disrupted by depleting the calcium, complete media with 0.1 µM or 1 µM ceramide was then added to measure re-assembly of TJ. After 24h the transepithelial electrical resistance (TEER) was measured and apical to basolateral permeability of sodium-fluorescein (MW 376 Da) which is used as an index of permeability (Papp). With 0.1 µM ceramide TEER was 138 ± 15 Ω.cm2 compared to control 125 ± 14 Ω.cm2 (p<0.01, n = 4). Papp was 16 ± 6 vs 18 ± 7 cm/s x 10-6, p= 0.19; n = 3, 0.1 µM ceramide vs control respectively. No changes were observed with 1 µM ceramide. Immunofluorescence staining for occludin, Z0-1 or claudin-3 did not show a difference in localisation with ceramide treatment compared to control cells. TJ were also disrupted by adding pro-inflammatory mediators (PIMS) -100 ng/ml TNF-α, 100 ng/ml IFN-γ and 10 μg/ml LPS, in the presence of ceramide. TEER, Papp and TJ immunofluorescence was measured after 24h, 48h and 72h. TEER was significantly reduced by PIMS at 24h and 48h but not at 72h (205 ± 31 Ω.cm2 vs 401 ± 95 Ω.cm2, p<0.01, n=4) and (188 ± 51 Ω.cm2 vs 413 ± 49 Ω.cm2, p<0.01, n=4) compared to control. Ceramide did not however increase PIMS induced decrease in TEER. Permeability with PIMS was significantly increased after 72h from 5.9 ± 2.5 cm/s x 10-6 to 13.4 ± 1.9 cm/s x 10-6, p<0.001, n=3. Ceramide + PIMS at 0.1 µM decreased Papp to 9.0 ± 2.2 (p<0.05, n=3) and at 1 µM to 6.8 ± 1.3 (p<0.01, n=3) both vs PIMS. Immunofluoresence staining for Z0-1 and claudin-3 was decreased with PIMS and restored with ceramide. These results indicate that in airway epithelial cells activation of PKCζ by ceramide only evokes a minor effect on tight junction reassembly or permeability. In the presence of PIMS ceramide does not change ionic resistance but does change permeability. This could be mediated through claudin-3 which can alter TJ meshwork and seals paracellular pathways against small ions and solutes5.