The effects of methylene blue, an inhibitor of soluble guanylate cyclase, on pulmonary vasodilator responses to efferent vagal stimulation were investigated in the intact-chest cat under conditions of controlled blood flow and constant left atrial pressure. In animals pretreated with reserpine or phenoxybenzamine, under elevated tone conditions, efferent vagal stimulation at frequencies of 2-16 Hz caused stimulus-frequency-dependent decreases in lobar arterial pressure and pulmonary lobar vascular resistance. The vasodilator response to vagal stimulation was reproducible, blocked by atropine, and reduced by methylene blue. Intralobar infusion of methylene blue increased lobar arterial pressure without significantly altering systemic arterial or left atrial pressure. Methylene blue had no significant effect on vasodilator responses to isoproterenol, albuterol, atriopeptin III, lemakalim, adenosine, ATP, and pituitary adenylate cyclase-activating polypeptide-27 but significantly decreased vasodilator responses to acetylcholine, nitric oxide (NO), sodium nitroprusside, and the S-nitrosothiol, S-nitroso-N-acetyl-penicillamine. The effects of methylene blue on responses to vagal stimulation were reversible and were similar with the addition of a NO synthase inhibitor. The present data suggest that vasodilator responses to cholinergic nerve stimulation involve an increase in the production of guanosine 3',5'-cyclic monophosphate in the pulmonary vascular bed. These results provide additional evidence to support the hypothesis that neurogenically released acetylcholine induces endothelium-dependent, muscarinic, guanylate cyclase-mediated vasodilation.