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Chronic nitroglycerin treatment results in development of nitrate tolerance associated with endothelial dysfunction (ED). We sought to clarify how mitochondria- and NADPH oxidase (Nox)-derived reactive oxygen species (ROS) contribute to nitrate tolerance and nitroglycerin-induced ED. Nitrate tolerance was induced by nitroglycerin infusion in male Wistar rats (100 μgh4 day) and in C57Bl6, p47phox−−and gp91phox−−mice (50 μgh4 day). Protein and mRNA expression of Nox subunits were unaltered by chronic nitroglycerin treatment. Oxidative stress was determined in vascular rings and mitochondrial fractions of nitroglycerin-treated animals by L-012 enhanced chemiluminescence, revealing a dominant role of mitochondria for nitrate tolerance development. Isometric tension studies revealed that genetic deletion or inhibition (apocynin, 0.35 mgh4 day) of Nox improved ED, whereas nitrate tolerance was unaltered. Vice versa, nitrate tolerance was attenuated by co-treatment with the respiratory chain complex I inhibitor rotenone (100 μgh4 day) or the mitochondrial permeability transition pore blocker cyclosporine A (50 μgh4 day). Both compounds improved ED, suggesting a link between mitochondrial and Nox-derived ROS. Mitochondrial respiratory chain-derived ROS are critical for the development of nitrate tolerance, whereas Nox-derived ROS mediate nitrate tolerance-associated ED. This suggests a crosstalk between mitochondrial and Nox-derived ROS with distinct mechanistic effects and sites for pharmacological intervention. [ABSTRACT FROM AUTHOR]