The amiloride-sensitive epithelial sodium channel (ENaC) is essential for fluid and electrolyte homeostasis. ENaC consists of alpha, beta, and gamma subunits, each of which contains a PPxY motif that interacts with the WW domains of the ubiquitin-protein ligases Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome in which mutations delete or alter the PPxY motif of either the beta or the gamma subunits, results in increased ENaC activity. We report here that Nedd4-2 has two major isoforms that show tissue-specific expression; however, both isoforms can inhibit ENaC in Xenopus oocytes. Because there are four WW domains in Nedd4-2, we analyzed binding kinetics and affinity between individual WW domains and ENaC subunits. Using whole cell patch-clamp techniques, we studied the role of individual WW domains in the regulation of ENaC in mammalian cells. We report here that unlike Nedd4, only two of the Nedd4-2 WW domains, WW3 and WW4, are required for both the binding to ENaC subunits and the regulation of Na+ feedback control of ENaC. Although both WW3 and WW4 individually can interact with all three ENaC subunits in vitro, both domains together are essential for in vivo function of Nedd4-2 in ENaC regulation. These data suggest that Nedd4-2 WW3 and WW4 interact with distinct, noninterchangeable sites in ENaC and that to prevent Na+ feedback control of ENaC it is necessary to occlude both sites.