Aims Nitroglycerin (or glyceryl trinitrate, GTN) has been long considered an endothelium-independent vasodilator because GTN vasodilation is intact in the absence of the endothelium and in the presence of endothelial dysfunction. However, in animal and in vitro models, GTN has been shown to stimulate the release of certain endothelium-derived vasodilators such as nitric oxide (NO) and prostacyclin (PGI2 ). In addition, chronic GTN therapy leads to endothelial dysfunction. In this series of experiments, we explored how GTN might interact with the vascular endothelium in normal humans, without cardiovascular disease or risk factors associated with abnormalities in vascular function. Methods We examined the effect of inhibition of NO, PGI2 , and epoxyeicosatrienoic acids (EETs, a class of endothelium-derived hyperpolarizing factor) on GTN-mediated vasodilation. We measured arterial blood flow responses to brachial artery infusions of GTN in the absence and presence of L-NMMA (n = 13), ketorolac (n = 14) and fluconazole (n = 16), which are inhibitors of endothelium-derived NO, PGI2 and EETs, respectively, in healthy volunteers. Results Our results demonstrate that inhibition of endothelium-dependent vasodilator mechanisms does not alter forearm resistance vessel responses to GTN. Conclusion We conclude that GTN-mediated dilation of forearm resistance vessels is largely independent of vascular endothelium.