The role of reactive oxygen species in the vascular pathology associated with atherosclerosis was examined by testing the hypothesis that impaired vascular reactivity results from the reaction of nitric oxide (·NO) with superoxide (O2/-), yielding the oxidant peroxynitrite (ONOO-). Contractility studies were performed on femoral arteries from rabbits fed a cholesterol-supplemented diet. Cholesterol feeding shifted the EC50 for acetylcholine (ACh)-induced relaxation and impaired the maximal response to ACh. We used pH-sensitive liposomes to deliver CuZn superoxide dismutase (SOD; superoxide:superoxide oxidoreductase, EC 22.214.171.124) to critical sites of ·NO reaction with O2/-. Intravenously injected liposomes (3000 units of SOD per ml) augmented ACh-induced relaxation in the cholesterol-fed group to a greater extent than in controls. Quantitative immunocytochemistry demonstrated enhanced distribution of SOD in both endothelial and vascular smooth muscle cells as well as in the extracellular matrix. SOD activity in vessel homogenates of liposome-treated rabbits was also increased. Incubation of β very low density lipoprotein with ONOO- resulted in the rapid formation of conjugated dienes and thiobarbituric acid-reactive substances. Our results suggest that the reaction of O2/- with ·NO is involved in the development of atherosclerotic disease by yielding a potent mediator of lipoprotein oxidation, as well as by limiting ·NO stimulation of vascular smooth muscle guanylate cyclase activity.