Endothelium-derived relaxing factor (EDRF), believed to be nitric oxide or a compound that releases nitric oxide, is a potent vasodilator produced by some arteries in response to acetylcholine (ACh) and bradykinin (BK). ACh and BK are potent dilators of perinatal pulmonary and systemic arteries. The objectives of this study were to determine if EDRF is present in newborn vessels and if EDRF mediates the vasodilator actions of ACh and BK. Arterial rings from newborn guinea pigs, 1 to 3 d old, were obtained from a branch of the main pulmonary artery and the descending aorta for isometric force bioassays. At their optimal resting tension, the rings were preconstricted with phenylephrine 10-5M in Krebs- Henseleit solution before adding incremental doses of ACh or BK. If the endothelium was intact, ACh (10s M) relaxed pulmonary arteries and aortas (64 ± 7%, 72 ± 9% relaxation, respectively, mean ± SE). ACh-induced relaxation (ACh 10-5M) in the pulmonary artery and aorta, respectively, was significantly (p<0.05) attenuated by 1) endothelial removal (11 ± 9%, 28 ± 10%) by rubbing the ring lumen; 2) methylene blue, 10-6M, (6 ± 8%, 7 ± 3%) that inhibits EDRF-associated cGMP production in smooth muscle; and 3) methemoglobin, 10-5M, (13 ± 9%, 17 ± 7%) that binds EDRF. The results for BK were similar to ACh for the pulmonary artery but BK did not relax the aorta. Indomethacin diminished relaxation of the pulmonary artery and aorta to the submaximal dose (10-5M) of ACh but indomethacin did not effect the relaxation to ACh 10-4M or BK. We conclude that EDRF is produced in the guinea pig pulmonary artery and descending aorta at birth and that EDRF is a mediator of the vasodilator actions of ACh and BK. Vasodilation by ACh may also involve activation of the cyclooxygenase pathway.