Proteases are involved in the processing and activation of the epithelial sodium channel (ENaC). The aim of the present study was to investigate whether the prototypical serine protease trypsin can activate ENaC in microdissected split open mouse renal distal tubules. Whole-cell patch clamp recordings from principal cells of connecting tubules (CNT) or cortical collecting ducts (CCD) demonstrated that addition of trypsin (20 µg/ml) to the bath solution increased the ENaC mediated amiloride-sensitive whole-cell current (I_Ami) in the majority of cells. In contrast, trypsin applied in the presence of an excess of soybean trypsin inhibitor had no stimulatory effect. The I_Ami response to trypsin was variable ranging from no apparent effect to a twofold increase in I_Ami with an average stimulatory effect of 31 % or 37 % in mice on low Na⁺ diet or on standard Na⁺ diet, respectively. In cultured M-1 mouse collecting duct cells a robust stimulatory effect of trypsin on I_Ami was only observed in cells pre-treated by protease inhibitors. This suggests that endogenous proteases contribute to ENaC activation in renal tubular cells and that the degree of ENaC pre-stimulation by endogenous proteases determines the magnitude of the stimulatory response to exogenous trypsin. In conclusion we provide electrophysiological evidence that trypsin can stimulate ENaC activity in native renal mouse tubules. Thus, in the kidney ENaC stimulation by extracellular proteases may be a relevant regulatory mechanism in vivo.