Endothelial dysfunction caused by increases in vascular oxidant stress that decrease bioavailable nitric oxide (NO) plays a critical role in the vascular pathobiology of hyperhomocysteinemia. Boosting cellular glutathione levels or increasing the activity of cellular glutathione peroxidase can compensate for homocysteine's effects on endothelial function. Aged garlic extract (AGE) contains water- and oil-soluble sulfur compounds that modify the intracellular thiol and redox state, minimize intracellular oxidant stress, and stimulate NO generation in endothelial cells and animals. We performed a placebo-controlled, blinded, crossover trial to examine whether AGE reduces macro- and microvascular endothelial dysfunction during acute hyperhomocysteinemia induced by an oral methionine challenge in healthy subjects. Acute hyperhomocysteinemia leads to a significant decrease in flow-mediated vasodilation of the brachial artery as determined by vascular ultrasound, indicative of macrovascular endothelial dysfunction. In addition, acute hyperhomocysteinemia leads to a decrease in acetylcholine-stimulated skin perfusion as measured by laser-Doppler flowmetry. This indicates microvascular endothelial dysfunction, which is presumably a result of impairment of the endothelium-derived hyperpolarizing factor pathway. Pretreatment with AGE for 6 wk significantly diminished the adverse effects of acute hyperhomocysteinemia in both vascular territories. We conclude that AGE may at least partly prevent a decrease in bioavailable NO and endothelium-derived hyperpolarizing factor during acute hyperhomocysteinemia. This pilot study warrants further investigations on the effects of AGE on endothelial dysfunction in patients with other cardiovascular risk factors or established vascular disease and on the clinical outcome of patients with cardiovascular disease.