Activity of the amiloride-sensitive Na+channel (ENaC) expressed in the distal nephron and respiratory epithelium has a profound impact on Na+ homeostasis, the regulation of blood pressure and respiratory surface fluid volume. Dysfunction of ENaC underlies human diseases such as Liddle’s syndrome, pseudohypoaldosteronism type 1 and cystic fibrosis. The activity of ENaC is tightly controlled by an array of physiological factors that exert their effect on transcription, trafficking and gating of the channel via multiple cellular signalling pathways. Hydrogen peroxide (H2O2) is one of the reactive oxygen species produced in response to oxidative stress, and it modulates a variety of physiological events. H2O2 exerts its effects via redox-sensitive proteins including transcription factors, protein tyrosine kinases and small G proteins. Previous studies suggested that H2O2 plays an important role in the regulation of ENaC, implicating it in the pathophysiology of pulmonary edema and acute lung injury. Consistent with this notion, exogenous H2O2 inhibits transepithelial ion transport and suppresses baseline expression of the α-subunit of ENaC in alveolar epithelial cells. In agreement with these reports, we found that H2O2 has a profound inhibitory effect on the amiloride-sensitive current in mouse collecting duct (M1) cells and on exogenous ENaC expressed in Fisher Rat Thyroid (FRT) cells. This inhibitory effect of H2O2 is attenuated in cells over-expressing a dominant-negative mutant of apoptosis signal-regulating kinase 1 (ASK-1), a ubiquitously expressed MAP kinase kinase kinase that is activated in response to cytotoxic stress. Our data further suggest that H2O2 inhibits the activity of ENaC via the ASK-1/p38 MAP kinase signalling cascade by a mechanism that interferes with proteolytic activation of ENaC subunits. H2O2 is also known to be an activator of the proto-oncogene tyrosine-protein kinase (Src), a non-receptor membrane-associated tyrosine kinase that plays an important role in cytoskeletal organization and cell proliferation, tyrosine phosphorylation of many cellular signalling molecules and is involved in the cellular signalling pathway by which endothelin-1 inhibits activity of ENaC. We found that Src mediates the inhibitory effect of platelet-derived growth factor (PDGF) on ENaC. Unlike ASK-1, Src alters total ENaC protein expression and the abundance of ENaC at the cell membrane, but has no effect on proteolytic cleavage of the channel subunits. The inhibitory effect of Src requires caveolin-1, suggesting that the effector molecules that are involved in the Src-mediated signalling system that regulates ENaC may be organised in the cholesterol-rich lipid raft sub-membrane domain.