In vitro and in vivo techniques were developed with genetically modified mice to determine whether endothelin-1 (ET-1) functions as an O2 mediator in closure of the ductus arteriosus (DA) at birth. Wild-type CD-1 and 129/SvEv mice with ETA receptor −/−, +/−, and +/+ genotypes were used. Isolated DA from term ETA +/+ fetuses contracted to O2 (5–95%) and a thromboxane A2 analog (ONO-11113, 0.1 μM). Instead, ET-1 elicited a dual response with weak relaxation (0.1 nM) preceding contraction (1–100 nM). Indomethacin (2.8 μM) was also a constrictor. ETA−/− DA, unlike ETA +/+ DA, contracted marginally to O2and ET-1 but responded to ONO-11113. O2 contraction was also reduced in ETA +/− DA. In vivo, DA constricted equally in tracheotomized ETA −/− and ETA +/+ newborns. Conversely, no DA constriction was seen in hyperoxic ETA −/− fetuses in utero, although it occurred in ETA+/+ and +/− littermates. We conclude that ET-1 mediates the DA constrictor response to O2. Without ET-1, however, the vessel still closes postnatally, conceivably caused by the withdrawal of relaxing influence(s).