Background: We investigated the locus of flow regulation of vascular tone in carotid arteries of C57 Bl/6 and eNOS<sup>–/–</sup> mice. Methods: Arterial segments (2–3 mm) were mounted in a perfusion myograph and preconstricted with 1 µ M phenylephrine before monitoring flow-induced changes in lumen diameter. Results: Both control and eNOS<sup>–/–</sup> 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. Conclusions: These findings indicate the existence of a previously unidentified endothelium-independent mechanism of flow-induced dilation in the carotid artery. Further investigations to identify the mechanisms that underlie this response may provide novel therapeutic directions in the treatment of disorders characterized by abnormal flow regulation of vascular tone.