The appropriate regulation of the epithelial sodium channel (ENaC) in the aldosterone-sensitive distal nephron is essential for the maintenance of body sodium balance and the long term regulation of arterial blood pressure. Recent evidence suggests that proteolytic processing of ENaC contributes to ENaC regulation. We investigated the effect of trypsin on transepithelial resistance (Rte ), transepithelial voltage (Vte ) and equivalent short circuit currents (Isc ) of M-1 mouse collecting duct cells grown on permeable support. In M-1 cells maintained under standard culture conditions application of trypsin to the apical side had little effect on baseline Isc but significantly increased Rte . Pretreatment of M-1 cells with aprotinin, a known inhibitor of extracellular serine proteases, reduced baseline Isc . Interestingly, application of trypsin to M-1 cells pre-treated with aprotinin caused a significant Isc increase which was sensitive to amiloride. Similarly, pre-treatment of the cells with a specific inhibitor of the intracellular protease furin reduced baseline Isc and revealed a substantial stimulatory effect of trypsin. A combination of furin inhibitor and aprotinin had a synergistic effect with an enhanced Isc response to trypsin. These findings indicate that intra- and extracellular proteases are likely to be important (patho)- physiological modulators of ENaC function in the kidney.