Nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) represent the two major endothelial autacoids involved in the local control of vascular tone. Here we describe a novel pathway leading to the calcium-independent activation of the endothelial NO synthase by shear stress and enhanced cellular tyrosine phosphorylation. In addition we present evidence that NO inhibits the formation of EDHF, which we have characterized as a transferable, beta-naphthoflavone-inducible P450-dependent metabolite of arachidonic acid.