The increase in O₂ tension after birth is a major factor stimulating ductus arteriosus (DA) constriction and closure. Here we studied the role of the mitochondrial electron transport chain (ETC) as sensor, H₂O₂ as mediator, and voltage-gated potassium (K_V) channels and Rho kinase as effectors of O₂-induced contraction in the chicken DA during fetal development. Switching from 0% to 21% O₂ contracted the pulmonary side of the mature DA (mature pDA) but had no effect in immature pDA and relaxed the aortic side of the mature DA (mature aDA). This contraction of the pDA was attenuated by inhibitors of the mitochondrial ETC and by the H₂O₂ scavenger polyethylene glycol (PEG)-catalase. Moreover, O₂ increased reactive oxygen species (ROS) production, measured with the fluorescent probes dihydroethidium and 2′,7′-dichlorofluorescein, only in mature pDA. The H₂O₂ analog t-butyl-hydroperoxide mimicked the responses to O₂ in the three vessels. In contrast to immature pDA cells, mature pDA cells exhibited high-amplitude O₂-sensitive potassium currents. The K_V channel blocker 4-aminopyridine prevented the current inhibition elicited by O₂. The L-type Ca²⁺ (Ca_L) channel blocker nifedipine and the Rho kinase inhibitors Y-27632 and hydroxyfasudil induced a similar relaxation when mature pDA were stimulated with O₂ or H₂O₂. Moreover, the sensitivity to these drugs increased with maturation. Our results indicate the presence of a common mechanism for O₂ sensing/signaling in mammalian and nonmammalian DA and favor the idea that, rather than a single mechanism, a parallel maturation of the sensor and effectors is critical for O₂ sensitivity appearance during development.