The endothelial nitric oxide (NO) synthase (eNOS, or NOS3) can be activated in response to fluid shear stress and numerous agonists via cellular events such as increased intracellular Ca2+, interaction with substrate, co-factors as well as adaptor and regulatory proteins, protein phosphorylation and S-glutathionylation in addition to shuttling between distinct sub-cellular domains. While some protein interactors modulate enzymatic activity, others can be S-nitrosation targets, which are brought in close proximity to the NO source under specific conditions. The identification of proteins interacting with eNOS in human endothelial cells stimulated with serum and growth factors may provide new insight into the regulation of eNOS activity as well as NO signaling via S-nitrosation.