Although neuronal nitric oxide synthase (nNOS) is involved in many physiological processes in skeletal muscle, nNOS knockout- mice (nNOS-/-) do not show any phenotypic malfunctions. Thus we searched for proteins with altered expression in extensor digitorum longus muscle (EDL) of nNOS-/- mice as compared to C57/Bl6-wild-type mice by 2D-PAGE. As identified by LC- MS/MS and immunoblotting, peroxiredoxin-6 was present only in nNOS-/- samples. Five other proteins up-regulated in the nNOS-/- mice were prohibitin, peroxiredoxin-3, superoxide dismutase (SOD1), heat shock protein beta-1 (HSP25) and nucleoside diphosphate kinase B (NDK-B). All six proteins influence the reactive oxygens species (ROS) metabolism. QRT-PCR showed mRNA up-regulation for four of these genes (exception: prohibitin, NDK-B). Intravital microscopy as well as determination of capillary density indicate the existence of a NOS- independent vasodilatory system in the EDL of nNOS-/- mice. Quantitative immunoblotting with antibodes against various vasodilatory enzymes revealed only a heme oxygenase (HO)-3- immunoreactive band to be upreguated in the nNOS-/- mice. Our data suggest that ROS metabolism and the HO3-immunoreactive protein contribute to nNOS compensation in the nNOS-/- mice.