Although the glycocalyx has been implicated in wall shear stress (WSS) mechanotransduction, the role of glycocalyx components in nitric oxide (NO) and reactive oxygen species (ROS) production remains unclear. Here, we tested the hypothesis that glycocalyx is implicated in both endothelial NO and O(2)(-) production. Specifically, we evaluated the role of hyaluronic acid (HA), heparan sulfate (HS), and sialic acid (SA) in NO and O(2)(-) mechanotransduction. Twenty-seven ex vivo porcine superficial femoral arteries were incubated with heparinase III, hyaluronidase, or neuraminidase, to remove HS, HA, or SA, respectively, from glycocalyx. The arteries were then subjected to steady-state flow and the effluent solution was measured for nitrites and the vessel diameter was tracked to quantify the degree of vasodilation. Our results show that removal of HA decreased both nitrites and vasodilation, and tempol treatment had no reversing effect. Degradation of HS proteoglycans decreased NO bioavailability through an increase in O(2)(-) production as indicated by fluorescent signals of dihydroethidium (DHE) and its area fraction (209+/-24% increase) and also removed extracellular O(2)(-) dismutase (ecSOD) (67+/-9% decrease). The removal of SA also increased O(2)(-) production as indicated by DHE fluorescent signals (86+/-17% increase) and the addition of tempol, a mimic O(2)(-) scavenger, restored both NO availability and vasodilation in both heparinase- and neuraminidase-treated vessels. This implies that HS and SA are not directly involved in WSS-mediated NO production. This study implicates HA in WSS-mediated NO mechanotransduction and underscores the role of HS and SA in ROS regulation in vessel walls in response to WSS stimulation.