The aryl hydrocarbon receptor-associated protein-9 (ARA9) is a chaperone of the aryl hydrocarbon receptor (AHR). The AHR has been shown to play a late developmental role in the normal closure of a fetal hepatovascular shunt known as the ductus venosus (DV). Given that Ara9-null mice display early embryonic lethality, we generated a hypomorphic Ara9 allele (designated Ara9(fxneo)) that displays reduced ARA9 protein expression. In an effort to demonstrate the role of ARA9 protein in AHR-mediated DV closure, we used combinations of Ara9 wild-type [Ara9(+/+)], null [Ara9(-/-)], and hypomorphic [Ara9(fxneo/fxneo)] alleles to produce mice with a graded expression of the ARA9 protein. Liver perfusion studies demonstrated that although none of the Ara9(+/+) mice displayed a patent DV, the shunt was observed in 10% of the Ara9(+/fxneo) mice, 55% of the Ara9(+/-) mice, and 83% of the Ara9(fxneo/fxneo) mice. That expression level of ARA9 correlates with the frequency of a phenocopy of the Ahr-null allele supports the conclusion that the ARA9 protein is essential for AHR signaling during development.