Extracellular superoxide dismutase type C (EC-SOD C) is a secretory SOD isoenzyme that, in vivo, is bound to heparan sulfate proteoglycans in the glycocalyx of various cell types (e.g., endothelial cells) and in the connective tissue matrix. The aim of this study was to investigate the efficacy of vascular bound EC-SOD C in protecting arterial relaxation mediated by endothelium-derived relaxing factor (EDRF) against the inhibitory effects of superoxide radicals. For comparison, the effect of CuZn SOD was also studied. This SOD isoenzyme lacks affinity toward heparan sulfate and does not bind to cell surfaces. Rings from rabbit aorta were mounted in an organ bath and acetylcholine-induced endothelium-dependent relaxation was then studied in preparations precontracted with phenylephrine. Pyrogallol (10−4M), used to generate superoxide radicals, reduced the maximal relaxant effect of acetylcholine from about 65% to 25%. When present in the buffer throughout the experiment, CuZn SOD and EC-SOD C caused a concentration-dependent prevention of the pyrogallol effect on EDRF-mediated relaxation, with a half-maximal effect at about 100 units/ml (KO2assay). In a second set of experiments, the arterial rings were preincubated with 8,000 units/ml CuZn SOD (50 μg/ml) or EC-SOD C (69 μg/ml) during 30 minutes, followed by washing, before the effect of pyrogallol on EDRF-mediated relaxation was studied in SOD-free buffer. The maximal relaxant effects of acetylcholine, expressed as the percent decrease in tension, were (mean±SEM,n=6) 61±4% (control), 22±4% (pyrogallol), 27±5% (pyrogallol+CuZn SOD), and 46±6% (pyrogallol+EC-SOD C;p<0.05 versus CuZn SOD+pyrogallol). The preincubation resulted in considerable binding of EC-SOD C to the arterial tissue, whereas negligible amounts of enzyme were present in the medium after the washing procedure. The protection by EC-SOD C was lost when the preincubation was performed in the presence of heparin (125 IU/mi), which prevents the binding of the enzyme to the arterial rings. In this case, the maximal relaxant effects of acetylcholine were (n=6) 60±4% (control), 21±4% (pyrogallol), 43±49% (pyrogallol+EC-SOD C), and 27±4% (pyrogallol+EC-SOD C+heparin;p<0.05 versus EC-SOD C+pyrogallol). It is concluded that EC-SOD C, associated with heparan sulfate proteoglycans on endothelial cell surfaces and internal structures in the aortic rings, protects against the detrimental effects of superoxide radicals on endothelium-dependent arterial relaxation.