In the aorta of the spontaneously hypertensive rat (SHR), endothelium-dependent contractions are enhanced by inhibitors of NO synthase and scavengers of NO, but not by methylene blue, an inhibitor of guanylyl cyclase, suggesting that the endothelium-derived contracting factor (EDCF) interacts chemically with NO and is inactivated by the latter. However, in view of the relative lack of specificity of methylene blue this hypothesis was re-examined. Acetylcholine-induced endothelium-dependent contractions of isolated rings of SHR aorta were significantly and similarly potentiated by two NOS inhibitors, by two structurally different NO scavengers, by two inhibitors of guanylate cyclase ODQ and NS2028, but to a lesser extent by methylene blue. The contraction of the isolated rat trachea in response to methacholine and the contraction of the rat aorta in response to both 8-isoprostane and KCl were inhibited significantly by methylene blue. Methylene blue binds to the M3 muscarinic receptor subtype but not to the TP receptor. Therefore, methylene blue is an antagonist of the M3 muscarinic receptor subtype, involved in the release of EDCF, and a non-specific inhibitor of TP receptor-mediated contractions, the receptor involved in the action of EDCF. These inhibitory effects of methylene blue are likely to counteract the effect of the inhibition of soluble guanylate cyclase. These results rule out the hypothesis according to which NO would chemically inactivate EDCF.