The epithelial sodium channel (ENaC) is the rate limiting step for sodium absorption in the aldosterone sensitive distal nephron. The appropriate regulation of renal ENaC activity is critically important for the maintenance of body sodium balance and hence for the long term regulation of arterial blood pressure. Proteolytic cleavage by intra- and extracellular proteases is thought to contribute to ENaC processing and regulation under physiological and pathophysiological conditions (Rossier 2004). Proteases acting from the extracellular side have been shown to activate near-silent channels in the plasma membrane (Caldwell et al. 2004). However, the underlying molecular mechanisms are not yet fully understood. We used trypsin, chymotrypsin and human neutrophil elastase (hNE) as tools to activate rat ENaC heterologously expressed in Xenopus laevis oocytes. Trypsin application increased ENaC whole-cell currents about 8-fold without a concomitant increase in channel surface expression. The stimulatory effect of trypsin was preserved in oocytes expressing αγENaC, but was abolished in oocytes expressing αβENaC. Thus, the γ subunit appears to be essential for channel activation by exogenous proteases. In single channel recordings from outside-out patches from oocytes expressing αβγENaC we demonstrated that trypsin (i) increases the open probability (P_O) of channels that are already active and (ii) activates a population of near-silent channels. This dual effect was confirmed by experiments using the β518C mutant ENaC which can be converted to a channel with a P_O of nearly one by exposure to a sulfhydryl reagent. Site-directed mutagenesis of a recently identified putative prostasin cleavage site in the extracellular loop of the γ subunit (Bruns et al. 2007) revealed that mutating the 181Lys residue to alanine (γK181A) largely reduces the stimulatory effect of extracellular proteases (trypsin, chymotrypsin and hNE), increases ENaC baseline whole-cell currents, and decreases channel surface expression. Moreover, the γK181A mutation promotes the spontaneous appearance of a 67 kDa fragment of the γ-subunit which also appears after channel activation by extracellular trypsin. At the single-channel level the γK181A mutation essentially abolishes the activation of near-silent channels by trypsin, while the stimulatory effect of trypsin on the P_O of active channels is preserved. We conclude that the putative prostasin cleavage site in the γ-subunit of ENaC is functionally important for the activation of near-silent channels by extracellular proteases.