Proteinuria, oedema and sodium retention are cardinal features of nephrotic syndrome. In the puromycin aminonucleoside (PAN) model of nephrotic syndrome in rats, a primary stimulation of the epithelial sodium channel (ENaC) in the late distal tubule and cortical collecting duct is involved in the sodium retention. However, the mechanism of this stimulation is unknown. We tested the hypothesis that the nephrotic urine itself has the capability of stimulating ENaC activity by using patch clamp experiments on single mouse cortical collecting cells that express ENaC. We found that nephrotic urine stimulated ENaC activity. Several serine proteases have the ability to stimulate ENaC activity. Consistent with this, nephrotic urine displayed serine protease activity and lost the ability to stimulate ENaC activity after pharmacological inhibition or affinity precipitation of serine proteases. To identify the candidate serine protease(s), we used a combination of aprotinin-affinity precipitation, ion exchange chromatography and MALDI-TOF mass spectrometry. An 80-kDa protein was isolated and identified as plasmin by mass spectrometry. Purified plasmin stimulated ENaC activity and addition of the plasmin inhibitors Pefabloc PL and a₂-antiplasmin to nephrotic urine abolished the serine protease activity and the ability to stimulate ENaC activity. Thus, our results suggest that plasmin is the dominant protease in nephrotic urine responsible for stimulation of ENaC activity.