Both extracellular and intracellular proteases can activate epithelial Na+ channels (ENaC). The mechanism by which serine proteases activate ENaC is unknown. We investigated the effect of the serine protease trypsin on in vitro translated and immunopurified α-, β-, and γ-rENaC subunits. Immunopurified subunit proteins were exposed to increasing concentrations of trypsin ranging from 0.002 to 2 μg/ml in Tris-buffered saline buffer for 2 h. The proteolytic mixture was subjected to SDS-PAGE and analyzed by autoradiography. Our results demonstrate that the β- and γ-subunits of ENaC were most susceptible to trypsin proteolysis, and exposure to as little as 0.002 μg/ml trypsin resulted in a reduction in the size of the β- and γ-transcripts by 7-8 kDa. By using N- and C-terminally truncated β- and γ-subunits, we determined that trypsin cleaved the C termini of both subunits, resulting in a channel structure resembling that seen in Liddle's disease. Exposure to 2 μg/ml trypsin completely digested all three subunits. Our results suggest different susceptibilities of proteolytic sites of ENaC subunits to trypsin. Thus, we propose that limited intracellular proteolysis may be one of the potential physiological mechanisms of sodium channel regulation.