The effects of N omega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of endothelium-derived relaxing factor (EDRF) production, on vascular tone and responses were investigated in the pulmonary vascular bed of the intact-chest cat under conditions of controlled blood flow and constant left atrial pressure. When pulmonary vascular tone was elevated with U-46619, intralobar injections of acetylcholine, bradykinin, sodium nitroprusside, isoproterenol, prostaglandin E1 (PGE1), lemakalim, and 8-bromo-guanosine 3',5'-cyclic monophosphate (8-bromo-cGMP) dilated the pulmonary vascular bed. Intravenous administration of L-NAME elevated lobar arterial and systemic arterial pressures without altering left atrial pressure. When U-46619 was infused after L-NAME to raise lobar arterial pressure to levels similar to those attained during the control period, vasodilator responses to acetylcholine and bradykinin were reduced significantly, whereas responses to PGE1, lemakalim, and 8-bromo-cGMP were not altered, and responses to nitroprusside were increased. There was a small effect on the response to the highest dose of isoproterenol, and pressor responses to BAY K 8644 and angiotensin II were not altered. These results are consistent with the hypothesis that EDRF production may involve the formation of nitric oxide or a nitroso compound from L-arginine and that EDRF production may have a role in the regulation of tone and in the mediation of responses to acetylcholine and bradykinin in the pulmonary vascular bed of the cat.