Recent studies demonstrate that oxidative inactivation of tetrahydrobiopterin (H₄B) may cause uncoupling of endothelial nitric oxide synthase (eNOS) to produce superoxide (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document}). H₄B was found recyclable from its oxidized form by dihydrofolate reductase (DHFR) in several cell types. Functionality of the endothelial DHFR, however, remains completely unknown. Here we present findings that specific inhibition of endothelial DHFR by RNA interference markedly reduced endothelial H₄B and nitric oxide (NO·) bioavailability. Furthermore, angiotensin II (100 nmol/liter for 24 h) caused a H₄B deficiency that was mediated by H₂O₂-dependent down-regulation of DHFR. This response was associated with a significant increase in endothelial \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} production, which was abolished by eNOS inhibitor N-nitro-l-arginine-methyl ester or H₂O₂ scavenger polyethylene glycol-conjugated catalase, strongly suggesting H₂O₂-dependent eNOS uncoupling. Rapid and transient activation of endothelial NAD(P)H oxidases was responsible for the initial burst production of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}}\end{equation*}\end{document} (Rac1 inhibitor NSC 23766 but not an N-nitro-l-arginine-methyl ester-attenuated ESR \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} signal at 30 min) in response to angiotensin II, preceding a second peak in \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} production at 24 h that predominantly depended on uncoupled eNOS. Overexpression of DHFR restored NO· production and diminished eNOS production of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{{\bullet}-}\end{equation*}\end{document} in angiotensin II-stimulated cells. In conclusion, these data represent evidence that DHFR is critical for H₄B and NO· bioavailability in the endothelium. Endothelial NAD(P)H oxidase-derived H₂O₂ down-regulates DHFR expression in response to angiotensin II, resulting in H₄B deficiency and uncoupling of eNOS. This signaling cascade may represent a universal mechanism underlying eNOS dysfunction under pathophysiological conditions associated with oxidant stress.