Responses to substance P were investigated in the pulmonary vascular bed of the cat with controlled pulmonary blood flow and constant left atrial pressure. Under baseline conditions, intralobar injections of substance P caused small, inconsistent reductions in lobar arterial pressure (AP) and significant reductions in mean systemic AP without affecting left atrial pressure. Decreases in lobar AP were significant and dose related when lobar vascular resistance was increased with U-46619, a thromboxane A2 mimetic. When compared with other vasodilator agents, the order of potency was substance P approximately bradykinin > pituitary adenylate cyclase activating polypeptide (PACAP) > acetylcholine (in nmol). Pulmonary vasodilator responses to substance P were unchanged by administration of atropine, glibenclamide, or sodium meclofenamate or when airflow to the left lower lung lobe was interrupted by bronchial occlusion. The NO synthesis inhibitor, N omega-nitro-L-argininemethyl ester (L-NAME), and the soluble guanylate cyclase inhibitor, methylene blue (MB), selectively inhibited pulmonary vasodilator responses to substance P and to acetylcholine. MB or L-NAME had no significant effect on pulmonary vasodilator responses to albuterol, lemakalim, or PACAP, whereas MB inhibited and L-NAME enhanced vasodilator responses to NO and sodium nitroprusside. The present investigation demonstrates that, when tone is increased experimentally, substance P has potent pulmonary vasodilator activity, and responses are not dependent on changes in bronchomotor tone, on the activation of muscarinic receptors or ATP-sensitive K+ channels, or on the release of a dilator prostaglandin but do involve, at least in part, endothelium-derived NO release and soluble guanylate cyclase activation.