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We investigated the locus of flow regulation of vascular tone in carotid arteries of C57 Bl/6 and eNOS–/–mice. Arterial segments (2–3 mm) were mounted in a perfusion myograph and preconstricted with 1 µM phenylephrine before monitoring flow-induced changes in lumen diameter. Both control and eNOS–/–mice demonstrated flow-dependent relaxation. This response was not attenuated by the NO synthase antagonist L-NAME, the cyclooxygenase inhibitor indomethacin, the selective guanylate cyclase inhibitor 1H-[1, 2, 4] oxadiazolo [4, 3-a] quinoxaline-1-one (ODQ), the adenylate cyclase inhibitor Rp-8-Br-cAMPs, integrin-binding RGD peptides, or by removal of the endothelium. Hypoxia, a physiological stimulus known to alter endothelium-dependent flow regulation of vascular tone, also failed to attenuate the observed flow-mediated dilation. These findings indicate …