The ability of human umbilical endothelial cells to release relaxing substance(s) in response to different agonists was investigated. Endothelium-denuded aortic rings of rats were used for the bioassay and tension recording. After precontraction, this preparation showed no response to histamine, acetylcholine, A 23187, or adenosine triphosphate while serotonin elicited further contraction. Super-fusion of the precontracted preparations with the perfusate from umbilical veins and arteries stimulated with histamine (10−7-10−5M), A23187 (10−7-10−6M), or adenosine triphosphate (10−5-10−4M) elicited a relaxation. No relaxation was obtained with acetylcholine (10−8-10−6M) or serotonin (10−8-10−6M). The relaxation of bioassay aortic rings under the influence of the perfusate from histamine-stimulated umbilical vessels was inhibited by mepyramine (10−5M) but not by cimetidine (10−4M) suggesting the involvement of H1-receptors. The relaxation was also inhibited by increasing the transit time between the donor and the detector preparation, by methylene blue (5 ± 10−5M), and by nordihydroguaiaretic acid (5 ± 10−5M) but not by indomethacin (5 ± 10−5M), characteristics which have been reported for endothelium-derived relaxing factor. The involvement of umbilical endothelial cells in the relaxation response was further confirmed by studying precontracted, rubbed rat aortic rings seeded with cultured endothelial cells from human umbilical veins. Such preparations relaxed in response to histamine (10−7-10−4M) in contrast with the control preparations. No relaxations of these preparations were observed in response to acetylcholine (10−9-10−6M). We conclude that the endothelial cells of the human umbilical blood vessels release an endothelium-derived relaxing factor in response to histamine, acting via H1-receptors, but not in response to acetylcholine.