Objective: Colon epithelium expresses epithelial Na⁺ channels (ENaC) and contributes to the fine regulation of gastrointestinal Na⁺ excretion under the control of corticosteroids. Proteolytic cleavage of ENaC subunits at extracellular sites has been shown to increase Na⁺ channel activity. In this study we investigated the role of this regulatory mechanism in rat distal colon in vitro. Methods: Rat distal colon epithelium was dissected from the muscular layer and investigated in a continuously perfused Ussing chamber (Ringer type solution, 23mM HCO₃^-, 5% CO₂, 37°C, 1M indomethacin). Cl^- secretion was blocked by azosemide (10mM). ENaC activity contributed to >=90% of the remaining electrogenic transport (effect of amiloride, 50mM). For long term observations, the antibiotic azlocillin (50mg/l) was added to the bath solution. Experiments were performed in open circuit mode and equivalent short circuit current I´sc was calculated from transepithelial voltage and resistance. Trypsin (10mg/ml) was added from the luminal side. Results: After equilibration, native tissue revealed a baseline current of -137±14mA/cm2 (n=8) which could not be increased by trypsin. Since membrane resident ENaC might have been already cleaved by luminal enzymes, we stimulated the additional luminal expression of ENaC by aldosterone in a long term experiment. While the baseline current showed a continuous rundown under control conditions, addition of aldosterone (0.3ng/ml) continuously increased I´sc after a lag time of 3h. At time 5.5h after the addition of aldosterone I´sc had increased to -368±79mA/cm2 (n=4), however, trypsin still did not affect I´sc significantly. Conclusion: ENaC in freshly isolated rat distal colon or additionally expressed ENaC after treatment with aldosterone can not be activated by proteolytic cleavage. Western blot analysis will show whether the respective subunits are resistant to cleavage or already cleaved during intracellular protein processing.