A hallmark of inflammation is increased vascular permeability. Increases in vascular permeability and the migration of inflammatory cells are linked to complex interactions of inflammatory mediators with the vascular endothelium. Normally, endothelial nitric oxide synthase (eNOS) produces a tonic amount of nitric oxide (NO), which is responsible for the homeostasis between the endothelium and surrounding tissues. However, most agonists that act on endothelial cells cause a series of post-translational modifications that influence eNOS activity. Furthermore, stimulation by shear stress, autacoids or growth factors either induces eNOS or shifts it to a more active state, which produces a burst of NO. Here, we highlight recent findings about eNOS and propose how new pharmacological tools can be used to dissect the involvement and contribution of eNOS to inflammatory responses.