ENaC is a heteromultimeric channel composed of 3 homologous subunits, α-,β- and γ-ENaC. It mediates the electrogenic Na⁺ transport across the apical membrane. The regulation of ENaC function is complex and involves various hormones such as aldosterone, vasopressin, insulin as well as epithelial growth factor (EGF). The acute mode of EGF-dependent inhibition of ENaC-mediated Na⁺ absorption is presumably by means of an increase in intracellular calcium and/or through the extracellular signal-regulated protein kinase (ERK) signaling pathway. The molecular mechanisms of long-term EGF-mediated regulation of ENaC function are not well known yet. To investigate the effects of the long-term application of EGF on ENaC function, we used a cystic fibrosis (CF) cell line, JME/CF15, derived from a nasal epithelium of a CF patient. Using the whole-cell mode of the patch-clamp technique, we measured an amiloride-sensitive Na⁺ current (2.0 ± 0.5 pA/pF at −100 mV; n=13) in cells growing in the EGF-free medium. This current was only observed in 1/3 of measured cells. The reversal potential of this current was dependent on Na⁺ concentrations and the cation selectivity was much higher for Na⁺ than for K⁺. These results indicate that the observed amiloride-sensitive Na⁺ current in JME/CF15 cells is most likely through ENaC channels. In contrast to cells growing in EGF-free conditions, cells growing in the EGF-containing medium did not show any amiloride-sensitive Na⁺ currents. This effect was transient, since amiloride-sensitive Na⁺ currents were measured again when the cells were shifted back to the EGF-free medium. Immunocytochemical experiments with specific anti-αENaC antibodies in permealized JME/CF15 cells showed that in cells cultured in EGF-containing medium α ENaC was mainly detected in a region around the nucleus, most likely in the endoplasmic reticulum (ER), while in cells growing in the EGF-free medium αENaC signals were more distributed over the cell body. In addition, the western blotting analysis of the whole cell extract from cells growing in the presence of EGF showed 2-fold increase of non-glycosylated and the absence of glycosylated αENaC fractions when compared to cells from the EGF-free culture. Taken together our data show that in JME/CF15 cells, the ENaC channel function is completely inhibited by the long-term incubation with EGF most likely as an effect of impaired trafficking and surface expression of ENaC channels.