Consistent with its classification as a Ca2+/calmodulin-dependent enzyme the constitutive endothelial nitric oxide (NO) synthase (eNOS) can be activated by receptor-dependent and -independent agonists as a consequence of an increase in the intracellular concentration of free Ca2+ ([Ca2+]i) and the association of the Ca2+/calmodulin complex with eNOS. Additional post-translational mechanisms regulate the activity of eNOS, including the interaction of eNOS with caveolin-1, heat shock protein 90 (Hsp90), or membrane phospholipids, as well as enzyme translocation and phosphorylation. In response to fluid shear stress the maintained production of NO by native and cultured endothelial cells is associated with only a transient increase in [Ca2+]i. In the absence of extracellular Ca2+ and in the presence of calmodulin antagonists, shear stress stimulates a maintained production of NO which is insensitive to the removal of extracellular Ca2+, but sensitive to tyrosine kinase inhibitors, Hsp90-binding proteins and phosphatidylinositol 3-kinase inhibitors. A pharmacologically identical activation of eNOS can be induced by protein tyrosine phosphatase inhibitors suggesting that the phosphorylation of eNOS, and possibly that of an associated regulatory protein(s), is crucial for its Ca(2+)-independent activation.