The amount of sodium and potassium 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 sodium and potassium in the regulation of two Pleckstrin Homology domain-containing intracellular signaling molecules - phospholipase C gamma-1 (PLC-1) and Epithelial and endothelial tyrosine kinase/Bone marrow tyrosine kinase on chromosome X (Bmx) - and agonist-generated calcium signaling in the endothelium. Extracellular [K⁺] regulated the levels of activated PLC-1, Bmx and carbachol-stimulated intracellular Ca²⁺ mobilization in human endothelial cells. Additional experiments confirmed that high-conductance calcium-activated potassium channels and phosphatidylinositol 3-kinase mediated these effects. The content of sodium and potassium in the diet also regulated Bmx levels in endothelial cell and activated PLC-1 levels in rats in vivo. The effects of dietary potassium on Bmx was more pronounced in rats on the high-salt diet, compared to rats on the low-salt diet. These studies elucidated an endothelial cell signaling mechanism regulated by electrolytes, further demonstrating an integral relationship between endothelial cell function and dietary sodium and potassium content.