Endothelium-dependent vasorelaxation in large vessels is mainly attributed to L-NAME-sensitive endothelial nitric oxide synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of nitric oxide synthases and cyclooxygenases. H₂O₂ has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta nNOS-derived H₂O₂ accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings ACh-induced relaxation was inhibited by N-Nitro-L-arginine methyl ester (L-NAME) and -Nitro-L-Arginine (L-NNA) two non-selective inhibitors of NOS and attenuated by selective inhibition of nNOS with L-Arg^NO2-L-Dbu-NH2 2TFA (L-Arg^NO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H₂O₂ production in endothelial cells that was prevented by addition of L-NAME, L-NNA, L-Arg^NO2-L-Dbu, TRIM and removal of the endothelium. Addition of catalase, an enzyme that degrades H₂O₂, reduced ACh-dependent relaxation and abolished ACh-induced H₂O₂ production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS, but not iNOS in mice aorta. Constitutive expression of nNOS was confirmed by Western blot analysis in endothelium containing- but not in endothelium denuded-vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Anti-sense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H₂O₂ production. Anti-sense knockdown of eNOS decresed ACh-induced relaxation but not H₂O₂ production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by selective inhibition of nNOS with L-Arg^NO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H₂O₂ is a major endothelium dependent relaxing factor. Hence, in the mouse aorta the effects of non-selective NOS inhibitors cannot be solely ascribed to NO release and action without considering the co-participation of H₂O₂ in mediating vasodilatation.