Phosphorylation of specific residues in the C-termini of the b- and [gamma]-subunit (bT613;[gamma]T623) of the epithelial sodium channel (ENaC) is thought to facilitate Nedd4 mediated channel retrieval (Shi et al. 2002 J Biol Chem 277: 13539). These sites have also been shown to contribute to ENaC regulation by progesterone (Michlig et al. 2005 J Biol Chem 280: 38264-70) and by cAMP (Yang et al. 2006 J Biol Chem 281: 9859) in the Xenopus laevis oocyte expression system. To directly confirm channel phosphorylation at the bT613 site, we used a phospho-specific antibody generated against a phosphopeptide corresponding to the region of interest. Using immunoprecipitation and Western blot analysis of HA-tagged bENaC we demonstrated that phosphorylation of bT613 was strongly enhanced by treatment with progesterone. In contrast, the phospho-specific antibody did not recognize a bT613A mutant channel confirming the specificity of the antibody. Interestingly, phosphorylation of bT613 was much stronger in oocytes maintained in high sodium bath solution compared to oocytes kept in low sodium bath solution to prevent sodium feedback inhibition. These findings suggest that phosphorylation of bT613 contributes to the inhibition of ENaC by progesterone as well as to sodium feedback inhibition of the channel most likely by enhancing its interaction with Nedd4.