The role of mitochondrial superoxide dismutase (Mn-SOD) in the maintenance of vascular function has not yet been studied. Thus, we examined flow and agonist-induced dilations in isolated mesenteric arteries (~90 µm in diameter) of Mn-SOD heterozygous (Mn-SOD^+/-) and wild-type (WT) mice. Increases in flow elicited dilations in all vessels, but the magnitude of the dilation was significantly less in vessels of Mn-SOD^+/- mice than in those of WT mice (64 vs 74% of passive diameter). L-NAME inhibited the dilation in vessels of WT mice but had no effect on vessels of Mn-SOD^+/- mice. Tempol or Tiron, superoxide scavengers, increased flow-induced dilation in vessels of Mn-SOD^+/- mice. Acetylcholine and sodium nitroprusside-induced, but not adenosine-induced, dilations were also decreased in arteries of Mn-SOD^+/- mice. Superoxide levels in arteries of Mn-SODm^+/- ice were significantly increased. Western blot analysis confirmed a 50% reduction of Mn-SOD protein in vessels of Mn-SOD^+/- mice. A 41% reduction in eNOS protein and a 37% reduction in eNOS activity were also found in vessels of Mn-SOD^+/- mice. While, there was no difference in eNOS protein in kidney homogenates of WT and Mn-SOD^+/- mice, a significant reduction of NOS activity was found in Mn-SOD^+/- mice, which could be restored by administration of Tiron. We conclude that an increased concentration of superoxide due to reduced activity of Mn-SOD-which inactivates NO and inhibits eNOS activity-contributes to the impaired vasodilator function of isolated mesenteric arteries of Mn-SOD^+/- mice. These results suggest that Mn-SOD contributes significantly to the regulation of vascular function.