We have previously reported the existence of multiple isoforms of human Nedd4-2 (AJP Renal 2003, 285: F916). When overexpressed in M-1 collecting duct epithelia, full length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the N-terminal C2 domain (Nedd4-2C2) and Nedd4-2 lacking WW domains 2 and 3 (Nedd4-2WW2,3) variably reduce benzamil-sensitive Na⁺ transport. We investigated the effect of each of the Nedd4-2 isoforms on cell-surface expression and ubiquitination of ENaC subunits. We find that ENaC when transfected alone or with and ENaC is expressed at the cell surface and this membrane expression is variably reduced by co-expression with each of the Nedd4-2 isoforms. Nedd4-2 reduces the half life of ENaC subunits and enhances the ubiquitination of , and ENaC subunits when expressed alone or together suggesting that each subunit is a target for Nedd4-2 mediated ubiquitination. As has been reported recently, we confirm that the surface expressed pool of ENaC is multi-ubiquitinated. Inhibitors of the proteasome increase ubiquitination of ENaC subunits and stimulate Na⁺ transport in M-1 cells consistent with a role for the ubiquitin-proteasome pathway in regulating Na⁺ transport in the collecting duct.