A superfusion cascade system was used to determine whether endothelium-dependent vasorelaxation induced by acetylcholine (ACh) is mediated by a humoral factor(s) in the domestic fowl. An abdominal aorta (5 cm in length) with intact endothelium, excised from the donor bird, was mounted in an organ bath and perfused with avian Ringer solution. The vasorelaxing activity of the effluent was determined by an endothelium-denuded aortic ring (bioassay ring) equilibrated and precontracted with phenylephrine (PHE) (10-6 M). The effluent (containing PHE) from the donor aorta with intact endothelium did not significantly change the PHE-induced tension of the bioassay rings. ACh (10-6 M), when added to the perfusion medium that runs through the control polyethylene tubing, produced further contraction of the PHE-precontracted endothelium-denuded bioassay rings, whereas ACh caused relaxation of the rings when added to the perfusate that passes through the endothelium-intact donor aorta. The ACh-induced relaxation of the bioassay ring was sightly potentiated by superoxide dismutase (100 U/ml). Hemoglobin (10-5 M) increased the basal tension of the bioassay rings and completely inhibited the ACh-induced relaxation of the rings. These results suggest that ACh-induced relaxation of fowl aorta is, at least partially, mediated by a humoral factor(s) that resembles endothelium-derived relaxing factor demonstrated in mammals. © 1991.