The epithelial sodium channel (ENaC) is the major rate-limiting step for vasopressin and aldosterone sensitive Na⁺ reabsorption across kidney epithelia. Recently, ENaC activity was shown to be modulated by extracellular factors such as proteases, Na⁺ ion and several other elements. However, the molecular mechanisms of these actions remain unclear. We and others have shown that ENaC composed of the guinea-pig α-subunit (αgp), and the β γ rat subunits (βrγr) could be activated by cpt-cAMP, a cAMP analogue, through a mechanism not involving the cAMP-PKA pathway. In the present study, we confirmed by patch-clamp experiments on Xenopus oocytes that the number of open channels increased by 2.4-fold after cpt-cAMP exposure. In order to characterize the extracellular domain involved in this activation, we generated α-subunit chimera's harboring different portions of the extracellular loop of the αgp and αr. Using two-electrode voltage-clamp, we established that Tyr456-Ser532 from the αgp confers sensibility to cpt-AMP. Then, by site-directed mutagenesis, we have isolated Ile481 as a major residue for cpt-cAMP-dependant activation. Taken together, these experiments provide evidence of an extracellular-ligand stimulating ENaC. They also contribute to the further understanding of the structure-function relationship of this channel.