Hypoxia augments alpha-adrenergic contraction (HVC) response was consistently observed in deep femoral arteries (DFA) of rats. We previously suggested that hypoxic inhibition of eNOS expressed in skeletal artery smooth muscle cells could be an important key on mechanism of HVC (Han et al., 2013). To investigate the role of muscular eNOS in skeletal arteries, we tested HVC response in femoral arteries (FAs) used wild type (WT) mice compared with eNOS hetero (HZ) and eNOS knockout (KO) mice. In analysis of immunohistochemistry, eNOS is expressed in medial layers of WT FAs as well as endothelium (n=12). However, the medial eNOS signal was not evident in HZ FAs (n=6), despite strong expression in the endothelium; similar observations were made in WT mesenteric arteries (MAs) (n=4) and carotid arteries (CAs) (n=5). Using myogrph, exposure to hypoxia (3% PO2) significantly augmented PhE-induced contraction in WT FAs unrelated to existence of endothelium (77 % of 80 mM KCl contraction, total n=86), but not in HZ (n=21) or KO (n=14) FAs. No HVC was observed in PhE-pretreated WT CAs also (n=7). The NOS inhibitor L-NAME (100 μM) also augmented PhE contraction in endothelium-denuded WT FAs (78 % of 80 mM KCl contraction, n=34), but not in HZ (n=21), KO (n=14) and WT CAs (n=7). Inhibitors specific to iNOS (1400W, 5 μM, n=9) and nNOS (SMTC 100 nM, n=6) did not prevent PhE-induced HVC contraction of WT FAs. NOX4 inhibitor (GKT137831, 5 μM, n=7, P < 0.001) but not NOX2 inhibitor (apocynin, 100 μM, n=4) suppressed HVC. Consistent with the role of reactive oxygen species, HVC was also inhibited by pretreatment with 352 U/ml PEG-catalase (n=5, P < 0.05) or 1 mM tiron (n=4, P < 0.05). 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 in a NOX4-ROS dependent mechanism, which potentiates vasoconstriction.