Epidermal growth factor (EGF) plays an important role in the regulation of cell growth, proliferation, and differentiation by binding to and activating the EGF receptor (EGFR). The EGFR is expressed by bronchial epithelial cells and regulates not only tissue repair, but also mucin production (1). EGFR signalling and potassium channel activity facilitate cell proliferation and migration. The inhibition of potassium channels by clofilium and glibenclamide reduced wound repair in NuLi (normal lung) and CuFi (CF) cells (2) and the EGFR is upregulated in the airways of asthma and COPD patients (3). While the effect of activation of EGF/EGFR in the gut is well documented (4), comparatively little is known about the role of the EGFR in ion transport in airway cells. Therefore, the aim of this study was to investigate the EGF signalling pathway and the regulation of potassium channels in the Calu-3 submucosal cell line. Calu-3 cells were grown to confluency on permeable supports at an air-liquid interface (ALI). Supports were incorporated into an Ussing chamber 7-15 days after establishment of the ALI and short circuit current (ISC) across the monolayer was measured. Data are presented as mean ± SEM (n = number of experiments) and were statistically analyzed using a one-way ANOVA with Tukey’s post hoc test. Pretreatment of confluent Calu-3 monolayers with 10ng/ml EGF to the basolateral side for 1 hour resulted in an increase in the starting ISC (24.8 ± 3.0µA cm-2, n=10, P<0.001) compared to 12.1 ± 0.7µA cm-2 (n=98) seen in untreated controls. Basolateral membrane permeabilisation abolished this effect. Pretreatment with 5 µM AG1478 (an EGFR inhibitor), resulted in a starting ISC of 5.2 ± 1.3µA cm-2 (n=5, P<0.001) significantly lower than EGF alone (24.8 ± 3.0µA cm-2, n=10). Preincubation with 50 µM wortmannin, a phosphoinositol-3-kinase (PI3K) inhibitor, resulted in a starting ISC of 5.7 ± 1.5µA cm-2 (n=4, P<0.001), while staurosporine (0.1 µM), chelerythrine chloride (10 µM) and rottlerin (5 µM) resulted in starting ISC of 5.3 ± 0.8µA cm-2 (n=4, P<0.001), 4.3 ± 0.9µA cm-2 (n=4, P<0.001), and 7.8 ± 0.8µA cm-2 (n=5, P<0.001), respectively, in all cases significantly lower than EGF alone (24.8 ± 3.0µA cm-2, n=10). Pretreatment with charybdotoxin (1 µM), iberiotoxin (10 nM) and chromanol 293B (10 µM) resulted in a starting ISC of 11.3 ± 1.2µA cm-2 (n=5, P<0.01), 4.3 ± 0.6µA cm-2 (n=4, P<0.001) and 10.1 ± 3.2µA cm-2 (n=5, P<0.01), respectively, significantly lower than with EGF alone (24.8 ± 3.0µA cm-2, n=8). In conclusion, these results show that pretreatment with EGF to the basolateral membrane increases chloride driving force through an increased ion transport via the KCa3.1 and KV7.1 potassium channels through the EGFR and a PI3K/PKCδ dependant signalling mechanism.