We tested the hypothesis that endothelium-dependent relaxation in the chicken ductus arteriosus (DA) is developmentally regulated. Isolated DA rings from 15-, 19- and 21-day-old (externally pipped) chicken embryos relaxed to acetylcholine (ACh). This relaxation was unaffected by indomethacin but impaired by endothelium removal, by the NO synthase inhibitor L-NAME, and by the soluble guanylate cyclase inhibitor ODQ, suggesting the involvement of NO. This NO production was confirmed with the fluorescent probe DAF-2DA. The combination of apamin and charybdotoxin with L-NAME produced a further inhibition of ACh-induced relaxation, suggesting the participation of a putative EDHF. In the 21-day DA, the relaxations induced by ACh and sodium nitroprusside (SNP) were markedly reduced and scanning electron microscopy demonstrated an irregular endothelial lining with protrusion and detachment of endothelial cells. The relaxations induced by BAY 41-2272 and 8-Br cGMP were not affected by age. When compared with 5%, lower (0%) and higher (21, 95%) O2 concentrations impaired ACh-induced relaxation. In summary, we found that ACh induces endothelium-dependent relaxation of the chicken DA and that NO and EDHF are involved in this response. During chicken DA closure, endothelial cells undergo morphologic and functional alterations that result in the lack of endothelium-dependent relaxation.