Liddle's syndrome is a form of inherited hypertension linked to mutations in the genes encoding the epithelial Na⁺ channel (ENaC). These mutations alter or delete PY motifs involved in protein–protein interactions with a ubiquitin‐protein ligase, Nedd4. Here we show that Na⁺ transporting cells, derived from mouse cortical collecting duct, express two Nedd4 proteins with different structural organization and characteristics of ENaC regulation: 1) the classical Nedd4 (herein referred to as Nedd4–1) containing one ammo‐terminal C2, three WW, and one HECT‐ubiquitin protein ligase domain and 2) a novel Nedd4 protein (Nedd4–2), homologous to Xenopus Nedd4 and comprising four WW, one HECT, yet lacking a C2 domain. Nedd4–2, but not Nedd4–1, inhibits ENaC activity when coexpressed in Xenopus oocytes and this property correlates with the ability to bind to ENaC, as only Nedd4–2 coimmunoprecipitates with ENaC. Furthermore, this interaction depends on the presence of at least one PY motif in the ENaC complex and on WW domains 3 and 4 in Nedd4–2. Thus, these results suggest that the novel suppressor protein Nedd4–2 is the regulator of ENaC and hence a potential susceptibility gene for arterial hypertension.—Kamynina, E., Debonneville, C., Bens, M., Vandewalle, A., Staub, O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na⁺ channel. . FASEB J. 15, 204–214 (2001)