Toll like receptor (TLR)4 is a pattern recognition receptor expressed in endothelial and other cells, responsible for the sensing of endotoxin and host derived ligands. Our group has shown previously that the absence of TLR4 is associated with reduced endothelial dependent vasodilator responses and left heart hypertrophy in animal models. However, the mechanism behind reduced endothelial cell function in TLR4(-/-) mice is not known. We have used en face confocal imaging of mesenteric arteries from mice deficient in the TLR4 receptor stained with dihydroethidium (DHE) to measure superoxide production. Using the isometric wire myograph, mesenteric artery vasodilator responses to acetylcholine and MnCl2 (a superoxide dismutase mimetic) were measured. Mesenteric arteries from TLR4(-/-) mice had a reduced endothelial dependent relaxant response and increased superoxide levels when stimulated with acetylcholine. Increased levels of superoxide, as detected by DHE staining, were seen in vessels from TLR4(-/-) mice, which were reduced to control levels in the presence of MnCl2. Our observations suggest that loss of TLR4 increases superoxide generation which reduces the biological activity of endothelial derived nitric oxide and thereby explains the endothelial dysfunction and associated cardiovascular phenotype in TLR4(-/-) mice. These data implicate a novel cardio-protective role for TLR4 in vascular homeostasis. (C) 2011 Elsevier Inc. All rights reserved.