Abstract: Kex2 protease is the prototype for a family of proteases responsible for endoproteolytic cleavage at multi-basic motifs in the eukaryotic secretory pathway. Here we demonstrate that potassium ion can act as a modulator of Kex2 activity with an apparent affinity of approximately 20 mm. Other monovalent cations (Li(+), Na(+), etc.) display similar effects, but affinities are all over 20-fold lower. Potassium ion binding stimulates turnover at physiologically relevant Lys-Arg cleavage sites but reduces turnover with at least one incorrect sequence. Furthermore, the mammalian Kex2 homolog furin displays similar effects. In contrast, the neuroendocrine homolog PC2 is inhibited by potassium ion with all substrates examined. The pre-steady-state behavior of Kex2 is also altered upon binding of potassium ion, with opposite effects on acylation and deacylation rates. These biochemical data indicate that potassium ion concentration may function as a regulator of processing protease specificity and activity in the eukaryotic secretory pathway, with such enzymes potentially encountering compartments high in potassium ion caused by the action of antiporters such as yeast NHX1 (VPS44) or the mammalian NHE7.