The human delta epithelial sodium channel (deltaENaC) subunit is related to the alpha-, beta-, and gammaENaC subunits that control salt homeostasis. DeltaENaC forms an amiloride-sensitive Na+ channel with the beta and gamma subunits. However, the in vivo function of deltaENaC is not known. To gain insight into the function of deltaENaC, a yeast two-hybrid screen of a human brain cDNA library was carried out using the C- and N-terminal domains of deltaENaC. A novel deltaENaC-interacting protein called Murr1 (mouse U2af1-rs1 region) was isolated in the C-terminal domain screen. Murr1 is a 21-kDa protein mutated in Bedlington terriers suffering from copper toxicosis. The interaction of Murr1 and deltaENaC was confirmed by glutathione S-transferase pulldown assay and coimmunoprecipitation. To test the functional significance of the interaction, Murr1 was coexpressed with deltabetagammaENaC in Xenopus oocytes. Murr1 inhibited amiloride-sensitive sodium current in a dose-dependent manner. In addition, deletion of the last 59 amino acids of deltaENaC abolished the inhibition. Murr1 also bound to the beta- and gammaENaC subunits and inhibited alphabetagammaENaC sodium current. Therefore, these results suggest that Murr1 is a novel regulator of ENaC.