The amount of Na⁺ and K⁺ in the diet promotes significant changes in endothelial cell function. In the present study, a series of in vitro and in vivo experiments determined the role of Na⁺ and K⁺ in the regulation of two pleckstrin homology domain-containing intracellular signaling molecules, phospholipase C (PLC)-γ₁ and epithelial and endothelial tyrosine kinase/bone marrow tyrosine kinase on chromosome X (Bmx), and agonist-generated Ca²⁺ signaling in the endothelium. Extracellular K⁺ concentration regulated the levels of activated PLC-γ₁, Bmx, and carbachol-stimulated intracellular Ca²⁺ mobilization in human endothelial cells. Additional experiments confirmed that high-conductance Ca²⁺-activated K⁺ channels and phosphatidylinositol 3-kinase mediated these effects. The content of Na⁺ and K⁺ in the diet also regulated Bmx levels in endothelial cells and activated PLC-γ₁ levels in rats in vivo. The effects of dietary K⁺ on Bmx were more pronounced in rats fed a high-salt diet compared with rats fed a low-salt diet. These experiments elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary Na⁺ and K⁺ content.