Although many laboratories have shown that dietary NaCl (salt) intake increases NO production in rodents and humans, the mechanism has not been uncovered. In the present study, pharmacological and dominant-negative strategies were used to show that feeding a formulated diet containing increased amounts of salt to young male Sprague-Dawley rats induced the formation of an endothelial cell-signaling complex that contained proline-rich tyrosine kinase 2, c-Src (also known as pp60c-src), and phosphatidylinositol 3-kinase. In the setting of a high-salt diet, proline-rich tyrosine kinase 2 served as the scaffold for c-Src-mediated phosphatidylinositol 3-kinase activation. Phosphatidylinositol 3-kinase was the upstream activator of protein kinase B (Akt), which was responsible for phosphorylation of the rat endothelial isoform of NO synthase at S1176 and thereby promoted the increase in NO production. The combined findings illustrated the crucial role for a proline-rich tyrosine kinase 2-signaling complex in the endothelial response to salt intake. © 2008 American Heart Association, Inc.