During normal pregnancy, uterine blood flow (UBF) is increased in association with elevations of endothelial nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) expression. Shear stress increases endothelial-derived NO production to reduce vasomotor tone. We hypothesized that decreasing in vivo UBF, and thus shear stress, will decrease NO and/or eNOS levels. In this experiment, one of the main uterine arteries of chronically instrumented late pregnant sheep (125 ± 1 days' gestation [mean ± SEM]; n = 15) was occluded for 24 h. Cardiovascular parameters (systemic and uterine arterial pressure, heart rate [HR], and ipsilateral and contralateral UBF) and NO₂/NO₃ (NO_x) levels were evaluated. Although UBF measured using Transonic flow probes was reduced unilaterally 41.5% ± 2.1%, uterine perfusion pressure only fell 12.2% ± 4.5%. Systemic arterial blood pressure and HR were unaltered. Using radioactive microspheres, ipsilateral UBF was reduced ∼28% during occlusion. The redistribution of UBF to other reproductive tissues suggests that collateral circulation develops in response to occlusion. Systemic arterial and uterine venous NO_x levels were reduced 22.1% ± 6.7% and 22.6% ± 7.6%, respectively, during occlusion. Treatment with microspheres produced an unexpected initial (∼2.5 h) increase in systemic arterial and uterine venous NO_x levels by 116% ± 30% and 97% ± 49%, respectively. Despite a decline in NO_x levels after 6 h, no significant differences versus preocclusion NO_x levels were detected by 24 h of occlusion in this experimental group. In contrast, NO_x, UBF, and uterine perfusion pressure levels unexpectedly failed to return to baseline values following release of occlusion. No differences in uterine artery eNOS expression were demonstrated by Western analysis from occlusion. Thus, our data suggest that shear stress may mediate in vivo vasomotor tone via production of NO_x.