The serine protease prostasin (CAP1/Prss8, channel-activating protease-1) is a confirmed in vitro and in vivo activator of the epithelial sodium channel ENaC. To test whether proteolytic activity or CAP1/Prss8 abundance itself are required for ENaC activation in the kidney, we studied animals either hetero- or homozygous mutant at serine 238 (S238A; Prss8(cat/+) and Prss8(cat/cat)), and renal tubule-specific CAP1/Prss8 knockout (Prss8(PaxLC1)) mice. When exposed to varying Na+-containing diets, no changes in Na+ and K+ handling and only minor changes in the expression of Na+ and K+ transporting protein were found in both models. Similarly, the alpha- or gamma ENaC subunit cleavage pattern did not differ from control mice. On standard and low Na+ diet, Prss8(cat/+) and Prss8(cat/cat) mice exhibited standard plasma aldosterone levels and unchanged amiloride-sensitive rectal potential difference indicating adapted ENaC activity. Upon Na+ deprivation, mice lacking the renal CAP1/Prss8 expression (Prss8(PaxLC1)) exhibit significantly decreased plasma aldosterone and lower K+ levels but compensate by showing significantly higher plasma renin activity. Our data clearly demonstrated that the catalytic activity of CAP1/Prss8 is dispensable for proteolytic ENaC activation. CAP1/Prss8-deficiency uncoupled ENaC activation from its aldosterone dependence, but Na+ homeostasis is maintained through alternative pathways.