The hemodynamic and anti-ischemic effects of nitroglycerin (NTG) are rapidly blunted as a result of the development of nitrate tolerance. With initiation of NTG therapy, it is possible to detect neurohormonal activation and intravascular volume expansion. These so-called pseudotolerance mechanisms may compromise the vasodilatory effects of NTG. Long-term nitrate treatment also is associated with decreased vascular responsiveness caused by changes in intrinsic mechanisms of the tolerant vasculature itself. According to the oxidative stress concept, increased vascular superoxide (O2-) production and an increased sensitivity to vasoconstrictors secondary to activation of protein kinase C contribute to the development of tolerance. Nicotinamide adenine dinucleotide phosphate oxidase and the uncoupled endothelial nitric oxide synthase may be O2- -producing enzymes. Nitric oxide (NO) and O2-, both derived from NTG and the vessel wall, form peroxynitrite in a diffusion-limited rapid reaction. Peroxynitrite, O2-, or both may be responsible for the development of nitrate tolerance and cross-tolerance to direct NO donors (eg, sodium nitroprusside, sydnonimines) and endothelium-dependent NO synthase-activating vasodilators. Hydralazine is an efficient reactive oxygen species (ROS) scavenger and an inhibitor of O2- generation. When given concomitantly with NTG, hydralazine prevents the development of nitrate tolerance and normalizes endogenous rates of vascular O2- production. Recent experimental work has defined new tolerance mechanisms, including inhibition of the enzyme that bioactivates NTG (ie, mitochondrial aldehyde dehydrogenase isoform 2 [ALDH2]) and mitochondria as potential sources of ROS. NTG-induced ROS inhibit the bioactivation of NTG by ALDH2. Both mechanisms increase oxidative stress and impair NTG bioactivation, and now converge at the level of ALDH2 to support a new theory for NTG tolerance and NTG-induced endothelial dysfunction. The consequences of these processes for NTG downstream targets (eg, soluble guanylyl cyclase, cyclic guanosine monophosphate-dependent protein kinase), toxic effects contributing to endothelial dysfunction (eg, prostacyclin synthase inhibition) and novel applications of the antioxidant properties of hydralazine are discussed.