Nitric oxide (NO) and reactive oxygen species (ROS) have fundamentally important roles in the regulation of vascular tone and remodeling. While arterial disease and endothelial dysfunction alter NO and ROS levels to impact vasodilation and vascular structure, direct measurements of these reactive species under in vivo conditions with flow alterations are unavailable. In this study, in vivo measurements of NO and H₂O₂ were made on mesenteric arteries to determine if antioxidant therapies could restore normal NO production in SHR. Flow was altered from ~50-200% of control in anesthetized WKY and SHR by selective placement of microvascular clamps on adjacent arteries while NO and H₂O₂ were directly measured with microelectrodes. Relative to WKY, SHR had significantly increased baseline NO and H₂O₂ concentration (2572 ± 241 vs. 1059 ± 160 nM, p< 0.01; and 26 ± 7 vs. 7 ± 1 µM, p< 0.05, respectively). With flow elevation, H₂O₂, but not NO increased in SHR; NO but not H₂O₂ was elevated in WKY. Apocynin and PEG-catalase decreased baseline SHR NO and H₂O₂ to WKY levels and restored flow-mediated NO production. Suppression of NAD(P)H oxidase with gp91 ds-tat decreased SHR H₂O₂ to WKY levels. Addition of topical H₂O₂ to increase peroxide to the basal concentration measured in SHR elevated WKY NO to levels observed in SHR. The results support the hypothesis that increased vascular peroxide in SHR is primarily derived from NAD(P)H oxidase and increases NO concentration to levels that cannot be further elevated with increased flow. Short-term and even acute administration of anti-oxidants are able to restore normal flow-mediated NO signaling in young SHR.