We previously reported that hypoxia augments alpha-adrenergic contraction (HVC) of skeletal arteries in rats. The underlying mechanism may involve hypoxic inhibition of eNOS expressed in skeletal arterial myocytes (14). To further explore the novel role of muscular eNOS in the skeletal artery, we compared HVC in femoral arteries (FAs) from eNOS knockout (KO) mice with that from wild-type (WT) and heterozygote (HZ) mice. Immunohistochemical assays revealed that, in addition to endothelia, eNOS is expressed in the FA medial layer, albeit at a much lower level. However, the medial eNOS signal was not evident in HZ FAs, despite strong expression in the endothelium; similar observations were made in WT carotid arteries (CAs). The amplitude of contraction induced by 1 µM phenylephrine (PhE) was greater in HZ than in WT FAs. Hypoxia (3% PO2) significantly augmented PhE-induced contraction in WT FAs, but not in HZ or KO FAs. No HVC was observed in PhE-pretreated WT CAs. The NOS inhibitor L-NAME (0.1 mM) also augmented PhE contraction in endothelium-denuded WT FAs, but not in WT CAs. Inhibitors specific to nNOS and iNOS did not augment PhE-induced contraction of WT FAs. NOX4 inhibitor (GKT137831, 5 μM) but not NOX2 inhibitor (apocynin, 100 μM) suppressed HVC. Consistent with the role of reactive oxygen species, HVC was also inhibited by pretreatment with tiron or PEG-catalase. Taken together, these data suggest that the eNOS expressed in smooth muscle cells in FAs attenuates alpha-adrenergic vasoconstriction; this suppression is alleviated under hypoxia, which potentiates vasoconstriction in a NOX4/ROS-dependent mechanism.