Endothelial vasomotor function decreases with increasing age. Extracellular superoxide dismutase (ecSOD) protects against vascular dysfunction in several disease states. The purpose of this study was to determine whether endogenous ecSOD protects against endothelial dysfunction in old mice. Vasomotor function of aorta was studied ex vivo in wild-type and ecSOD^-/- mice at 11 or 29 months of age. Maximal relaxation to acetylcholine (10^-4) was impaired in adult (11 months) ecSOD-deficient mice (75±3%, mean±SE) compared to wild-type vessels (89±2%), (p<0.05). Maximal relaxation to acetylcholine (10^-4) was profoundly impaired in old (29 months) ecSOD-deficient mice (45±5%) compared to aorta from wild-type mice (75±4%), (p<0.05). There was a significant correlation between expression of ecSOD and maximal relaxation to acetylcholine, in adult and old mice. Tempol (1 mM), a scavenger of superoxide, improved relaxation in response to acetylcholine (63±8%) in old ecSOD-KO mice (p<0.05), but not in wild-type mice (75±4%). Maximal relaxation to sodium nitroprusside was similar in aorta from adult and old wild-type and ecSOD-deficient mice. In aorta of old mice, mRNA levels of ecSOD and catalase by quantitative RT-PCR were decreased, while levels of TNF and Nox-4 tended to be increased, compared to adult mice. The findings support the hypothesis that impaired antioxidant mechanisms may contribute to cumulative increases in oxidative stress and impaired endothelial function in old mice. In conclusion, endogenous ecSOD plays an important role in protection against endothelial dysfunction during aging.