Methylene blue has been frequently used as an inhibitor of soluble guanylyl cyclase. We found that endothelium-dependent relaxations of isolated blood vessels were considerably more sensitive to inhibition by methylene blue than relaxation induced by direct activators of soluble guanylyl cyclase. Similar data were obtained in the presence of superoxide dismutase, indicating that the diverse potencies of methylene blue were not due to superoxide-induced inactivation of nitric oxide (NO). Subsequent experiments revealed that methylene blue is an inhibitor of purified NO synthase. Conversion of L-arginine to L-citrulline was inhibited by the dye in a concentration-dependent fashion with half-maximal effects observed at 5.3 microM and 9.2 microM in the absence and presence of superoxide dismutase, respectively. Purified soluble guanylyl cyclase, however, was far less sensitive to methylene blue. When the enzyme was maximally stimulated with S-nitroso-glutathione, cyclic guanosine monophosphate,(cGMP) formation was reduced by 50% at approximately 60 microM methylene blue; 1 mM produced maximal inhibitions of about 70%. Our data indicate that methylene blue is only a poor inhibitor of soluble guanylyl cyclase. The dye seems to act primarily via inhibition of NO synthase, with enzyme-bound heme being a possible target in its inhibitory action.