Endothelium-dependent vasorelaxation in large vessels is mainly attributed to N^ω-nitro-l-arginine methyl ester (l-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H₂O₂ has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H₂O₂ accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by l-NAME and N^ω-nitro-l-arginine (l-NNA), two nonselective 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 the addition of l-NAME, l-NNA, L-Arg^NO2-L-Dbu, TRIM, and removal of the endothelium. The 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 inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H₂O₂ production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H₂O₂ production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the 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 nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H₂O₂ in mediating vasodilatation.