Na+ movement across various transporting epithelia depends on its entry through apical epithelial Na+ channels (ENaC) and its exit by means of a basolateral Na+-K+-ATPase. ENaC activity is inhibited by extracellular and intracellular Na+, helping maintain Na+ homeostasis through negative feedback. Noting that ENaC is activated by proteolytic cleavage, and is present in both cleaved and uncleaved forms at the cell surface, Knight et al. investigated Na+’s effects on its proteolytic activation. When human embryonic kidney (HEK) 293T cells expressing the α, β, and γ subunits of ENaC were incubated in medium containing 135 mM Na+, more of the cell surface α-subunit (αENaC) was in the full-length than in the cleaved form, whereas this ratio was reversed in 0 mM Na+. The ratio of full-length to cleaved αENaC increased as Na+ concentration was increased to 34 mM, but not thereafter. When Fischer rat thyroid cells expressing ENaC were used to create a barrier between compartments containing 135 mM Na+, the initial current sensitive to amiloride (an ENaC blocker) was greater in cells preincubated in 0 mM Na+ than in 135 mM Na+. Moreover, trypsin increased current in cells preincubated in 135 mM Na+ but had little effect on current in those preincubated in 0 mM Na+. A combination of pharmacological and mutational analysis indicated that proteolytic cleavage of ENaC was regulated by intracellular Na+. The 135 mM Na+ inhibited proteolysis of ENaC by trypsin without inhibiting trypsin’s enzymatic activity. The authors propose that increased intracellular Na+ inhibits ENaC activation through Na+-dependent regulation of cleavage site accessibility.