The epithelial sodium channel (ENaC) is a multimeric protein which may be formed by a combination of up to four homologous subunits named a-, ß-, g- and d-ENaC. Predominantly, the a-subunit is expressed in epithelia such as kidney, lung, and colon and there it usually forms functional channels with the ß- and d-subunit. The d-subunit was first found in human brain and kidney but the expression was also detected in human cell lines of lung, pancreatic and colonic origin. When co-expressed with ß and g accessory subunits in heterologous systems the two known isoforms of the d-ENaC subunit (d1 and d2) can build functional amiloride-sensitive Na⁺ channels. In the present study we investigated the expression of the d-subunit in human nasal epithelium. We cloned and sequenced the full-length cDNA of the ENaC d-subunit and showed that in nasal tissue at least isoform 1 is expressed. Furthermore, we carried out Western blot and immunofluorescence analyses to study the expression of the d-ENaC in human nasal epithelium. Additionally, we showed a functional expression of the d-ENaC subunit with electrophysiological measurements in modified Ussing chambers. Thereby, we demonstrated that Evans Blue, a d-subunit-specific antagonist, inhibits the activity of d-ENaC in human nasal epithelium. Furthermore, we downregulated d-ENaC expression with siRNA to verify the specificity of Evans Blue and thus the contribution of the subunit to native Na⁺-transport across epithelia. These findings raise the question if the d-subunit possesses important regulatory function and if it interacts with other ENaC subunits or members of the DEG/ENaC family in the human respiratory epithelium.