In a perfusion-superfusion system, an endothelium-free ring of rabbit thoracic aorta precontracted with phenylephrine was used to bioassay relaxing activity. As previously reported, when NaNO2 (1 mM) was added to the standard Krebs' solution (37 degrees C oxygenated, containing 30 microM EDTA), irradiation of the solution with long wavelength ultraviolet light, while it was perfusing through a glass tube proximal to the bioassay ring, resulted in a small degree of relaxation of the ring, and this relaxation was markedly potentiated when superoxide dismutase (SOD, 10 U/ml) was also present in the solution. The full potentiation by SOD of the relaxation produced by photoactivation of NO2- was matched by cytochrome c (30 microM), MnCl2 (30 microM) and CuCl2 (100 microM), all of which are scavengers of superoxide (O2-). No potentiation was produced by catalase (1,000 U/ml), ZnCl2 (100 microM) and boiled SOD. Also, CuCl2, when its concentration was no greater than that of the EDTA in the Krebs' solution, failed to potentiate the relaxation. These results are consistent with our earlier conclusion that the relaxant produced by irradiation of NO2- is NO and that it is normally inactivated rapidly by O2- also present in the irradiated perfusion fluid. When EDTA was omitted from the perfusing Krebs' solution, photoactivation of NO2- in the solution produced a very large relaxation of the bioassay ring, which was not enhanced by addition of SOD or other O2- scavengers.(ABSTRACT TRUNCATED AT 250 WORDS)