Recently we reported (Yang et al. 2005 JASN 16: 62A) that stimulation of the rat epithelial sodium channel (ENaC) by cAMP involves putative ERK phosphorylation sites in the C-termini of the channel's b- (T613A) and [gamma]-subunit (T623A). Our results suggested that an increase in intracellular cAMP favours the dephosphorylation of the two ERK sites which prevents channel retrieval and increases Po by reducing ENaC/Nedd4-2 interaction. We hypothesized that this mechanism acts synergistically with the previously reported mechanism that PKA mediated phosphorylation of Nedd4-2 inhibits ENaC/Nedd4-2 interaction (Snyder et al. 2004 J Biol Chem 279: 45753). Using co-expression studies in Xenopus laevis oocytes we demonstrated that mutating the two ERK sites reduced the inhibitory effect of wild type Nedd4-2 on ENaC currents (DI_Ami ). Coexpression of wild type Nedd4-2 did not prevent the stimulation of DI_Ami by a 24 h exposure to IBMX/forskolin. In contrast, coexpression of Nedd4-2 with mutated PKA sites (S328A, S222A) largely reduced the stimulatory effect of IBMX/forskolin. We conclude that stimulation of ENaC by cAMP involves both, dephosphorylation of ENaC and phosphorylation of Nedd4-2 at specific sites. This dual mechanism causes synergistic inhibition of ENaC/Nedd4-2 interaction resulting in ENaC stimulation.