Vascular non-leukocyte-derived reactive oxygen species (ROS), such as superoxide and hydrogen peroxide (H(2)O(2)), have emerged as important molecules in diabetic endothelial dysfunction. In addition, leukocyte-derived myeloperoxidase (MPO) has been implicated in vascular injury, and its injury response is H(2)O(2) dependent. It is well known that MPO can use leukocyte-derived H(2)O(2); however, it is unknown whether the vascular-bound MPO can use high-glucose-stimulated, vascular non-leukocyte-derived H(2)O(2) to induce diabetic endothelial dysfunction. In the present study, we demonstrated that MPO activity is increased in vessels from diabetic rats. In high-glucose-incubated rat aortas and in carotid arteries from rats with acute hyperglycemia, vascular-bound MPO utilized high-glucose-stimulated H(2)O(2) to amplify the ROS-induced impairment of endothelium-dependent relaxation via reduction of nitric oxide bioavailability. Hypochlorous acid (HOCL)-modified LDL, a specific biomarker for the MPO/HOCL/chlorinating species pathway, was detected in LDL- and MPO-bound vessels with high-glucose-stimulated H(2)O(2). The results suggest that vascular-bound MPO could use high-glucose-stimulated H(2)O(2) to amplify high-glucose-induced injury in the vascular wall. MPO/H(2)O(2)/HOCL/chlorinating species may represent an important pathway in diabetes complications and a new mechanism in phagocyte- and systemic infection-induced exacerbation of diabetic vascular diseases.