Objective: With the ability to inhibit the protein expression, antisense oligonucleotides (AON) offer new perspectives for the treatment of cystic fibrosis (CF). This disease is characterised by a defective chloride secretion mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) and excessively increased Na⁺ absorption via the amiloride-sensitive epithelial Na⁺ channel (ENaC). By downregulation of the ENaC mediated Na⁺ hyperabsorption in CF, AON could circumvent one of these basic problems of this severe disease. Therefore, the objective of this investigation was to find an effective inhibitor of human ENaC with respect to improve the therapy for CF patients. Therefore, we selected specific AON that efficiently suppress the Na⁺ hyperabsorption by inhibiting the expression of the a-ENaC subunit. Methods: For this study we used primary cultured human nasal epithelia cells derived from CF and non-CF tissue, which were transfected with AON. To prove the successful inhibition of ENaC expression we used Ussing chambers for functional studies and carried out biochemical as well as fluorescence optical analyses. Results: Using these methods we showed that AON repress effectively the amiloride-sensitive Na⁺ absorption mediated by ENaC in CF and non-CF tissues. With functional Ussing chamber studies we showed that the AON transfection inhibits the ENaC current by about 75% in CF and by about 66% in non-CF cells. Furthermore, we demonstrated that the AON are able to sustain this functional ENaC inhibition over a time period of 72 h. We verified this downregulation of the ENaC protein expression in Western Blot experiments and found a 46% reduction of the a-ENaC protein in AON transfected cells. Conclusion: Our data are a first step towards a preclinical AON test to reduce Na⁺ hyperabsorption in CF epithelia. From these data we conclude that ENaC specific AON could evoke a long-lasting repression of the excessive Na⁺ absorption in CF patients.