Nitric oxide (NO) produced by endothelium in response to vasorelaxants, such as acetylcholine, induces vasorelaxation of vascular smooth muscle cells (VSMC). It has been found that VSMC express NO-synthase, however, the principal question remained unanswered, if it is physiologically relevant. Because injury of endothelium triggers free-radical production which decreases NO availability we hypothesized that the destruction of arterial anatomical integrity by rubbing off endothelial layer made vessels insensitive to vasodilators as a consequence of oxidative stress. We examined acetylcholine-induced vasorelaxation in deendothelized thoracic aorta (TA), mesenteric artery (MA) and pulmonary artery (PA) of Wistar rats under protection against oxidative stress. Acetylcholine produced vasorelaxation in arteries with intact endothelium, whereas the relaxation in endothelium-denuded rings was inhibited. Pretreatment of TA, MA and PA denuded rings with tempol, free-radical scavenger, improved relaxation to acetylcholine compared to untreated rings. The improved relaxation in all denuded rings was inhibited when ODQ, an inhibitor of guanylate cyclase, or L-NAME, an inhibitor of NO-synthase, were administered contemporary with tempol. Chemiluminiscence method revealed that endothelial denudation of TA and PA increased the production of superoxides. Immunohistochemical staining confirmed expression of NOS3-isoform in both intimal and medial cells in all arteries. Results revealed that deendothelized arteries under protection against oxidative stress exerted relaxation to acetylcholine which was mediated by NO and cGMP. The study suggests that VSMC can release NO in amounts sufficient to account for the vasorelaxation. This finding challenges the concept of the exclusive role of endothelial cells in the relaxation of VSMC.