Chronic high blood flow and the hyperdynamic circulatory syndrome in portal hypertension are associated with endothelial constitutive nitric oxide (NO) synthase (eNOS) upregulation and increased NO release. In portal vein-ligated (PVL) rats the splanchnic circulation is not yet hyperdynamic on day 3 postoperatively. In vitro perfused superior mesenteric arteries (SMAs) of day 3PVL and sham rats were challenged with increasing flow rates or the α-adrenoreceptor agonist methoxamine (30 and 100 μM) before and after incubation with the NO inhibitor,N ^ω-nitro-l-arginine (l-NNA, 10⁻⁴ M). Perfusate NO metabolite (NO _x ) concentrations were measured by chemiluminescence. PVL rats expressed a significant hyporesponsiveness to increases in flow rate or methoxamine that was overcome by incubation with l-NNA. The PVL vasculature showed significantly higher slopes of NO _x production vs. flow-induced shear stress, higher increases in perfusate NO _x concentration in response to methoxamine, and higher eNOS protein levels (Western blot) compared with sham rats. In conclusion, eNOS-upregulation and increased NO release by the SMA endothelium occur before the development of the hyperdynamic splanchnic circulation, suggesting a primary role of NO in the pathogenesis of arterial vasodilatation.