Recent studies suggest that proteases like furin and prostasin can modify ENaC function and may play a role for ENaC regulation. However, the underlying mechanisms are not yet fully understood. In the present study we used trypsin as a tool to proteolytically activate rat ENaC expressed in Xenopus laevis oocytes. On average application of trypsin (2 mg/ml for 2 min) caused a 7.2-fold (n=91) increase in the amiloride-sensitive whole- cell current . This current increase was not accompanied by an increase in channel surface expression which was measured by a chemiluminescence assay using flag-tagged ENaC. In outside-out single channel patch clamp recordings we demonstrated that trypsin increases the open probability of channels that are active in the patch. In addition it recruits and activates a population of near-silent channels. Site-directed mutagenesis of a newely identified putative prostasin cleavage site in the extracellular loop of the [gamma]-subunit revealed that the 181Lys residue is critical for the activation of ENaC by trypsin. Its mutation to an alanine largely reduced the stimulatory effect of trypsin on whole cell ENaC currents and prevented the activation of near-silent channels in outside-out patches. This novel regulatory pathway involving a putative prostasin cleavage site in the [gamma]-subunit of ENaC may be (patho)-physiologically important in vivo.