Vascular oxidative stress is the key mechanism involved in the age-related decline in endothelium-dependent dilatation (EDD). We tested the hypothesis that xanthine oxidase (XO), a major vascular source of reactive oxygen species, contributes to the impairment in EDD with ageing. At baseline, brachial artery flow-mediated dilatation (FMD) was 55% lower in older (n= 9, 64 ± 2 years, 8M/1F, mean ±s.e.m.) versus young (n= 9, 26 ± 1 years, 8M/1F) healthy adults (3.41 ± 0.44 versus 7.53 ± 0.67%, P < 0.001), whereas endothelium-independent dilatation (EID; sublingual nitroglycerin) did not differ between groups. Plasma oxidized low-density lipoprotein (oxi-LDL), a measure of systemic oxidative stress, was greater at baseline in the older subjects (58.3 ± 5.9 versus 46.8 ± 2.4 U l−1, P < 0.05) and inversely correlated with baseline FMD (r=− 0.54; P < 0.05). Acute administration of allopurinol, a competitive inhibitor of XO, reduced plasma uric acid concentrations similarly in both groups (P < 0.001), but did not affect FMD, EID, or oxi-LDL in either group. Vascular endothelial protein expression of XO (immunofluorescence) was not different in antecubital venous cells from the young and older subjects (0.56 ± 0.12 versus 0.68 ± 0.19 XO intensity/human umbilical vein endothelial cell intensity, P= 0.49). We conclude that XO does not contribute to oxidative stress-associated reductions in peripheral conduit artery EDD with ageing in humans, possibly due to an absence of age-associated up-regulation of endothelial XO.