The mechanisms contributing to sepsis vascular dysfunction are not well known. We tested the hypothesis that peroxynitrite scavenging ameliorates sepsis-induced macrovascular and microvascular dysfunction. Male Sprague-Dawley rats were killed 48 h after cecal ligation (n = 15) and puncture or sham procedure (n = 15). Their aortas and mesenteric vessels were mounted in organ baths for isometric tension recording. We studied contraction in resting vessels (norepinephrine 1 nM-10 μM and 10 nM-10 μM) and endothelium-dependent relaxation (acetylcholine, 10 nM-10 μM and 1 nM-10 μM) for aortas and microvessels, respectively. Vascular rings were preincubated for 30 min with the superoxide scavenger Cu-Zn-superoxide dismutase (SOD) (100 U/mL), the SOD mimetic and peroxynitrite scavenger tempol (10 M), the NO synthase inhibitor N-nitro-l-arginine methyl ester (10 M), or the peroxynitrite decomposition catalyst manganese tetrakis(4-N-methylpyridyl)porphyrin (MnTMPyP) (10 M). Fluorescence to 3-nitrotyrosine, oxidized dihydroethidium, and NOS2 was assessed in vascular tissue. Vascular NOS2, endothelial nitric oxide synthase (NOS1), NADPH-oxidase-1 (NOX-1), and SOD expression was analyzed by reverse transcription-polymerase chain reaction. Sepsis induced (i) in macrovessels, impairment of norepinephrine-induced contractions; (ii) in microvessels, impairment in norepinephrine-induced contractions and acetylcholine-induced relaxations; (iii) aortic and microvascular tissue increased reactivity to 3-nitrotyrosine, oxidized dihydroethidium, NOS2, and increased expression of NOS2, as well as increased expression of NOX-1 in microvascular tissue. Contractile responses in aortic and microvascular rings improved by ex vivo treatment with MnTMPyP and tempol, whereas vascular relaxation in microvessels improved only with MnTMPyP. Peroxynitrite scavenging protects from vascular dysfunction in sepsis.