Two major sources of ROS in vascular tissue are xanthine oxidase (XO), which generates ROS from the metabolites of adenosine and Nox¹. The vasodilatation that occurs in skeletal muscle during acute systemic hypoxia is largely mediated by adenosine and nitric oxide (NO)². During acute systemic hypoxia, ROS levels increase in skeletal muscle, and the hypoxia-induced muscle vasodilatation is modulated by XO-derived ROS which may act directly, or by interacting with NO³: O₂^– decreases NO bioavailability by combining with NO to form peroxynitrite. Nox activity is increased in chronic conditions such as hypertension⁴, but Nox expression was also increased by acute hypoxia in pulmonary artery⁵. It is not known whether ROS generated acutely from Nox can modulate skeletal muscle dilatation. Thus, in anaesthetized male Wistar rats (Alfaxan: 3-6ml.kg^-1.hr^-1 iv), arterial blood pressure (ABP) and femoral blood flow (FBF) were recorded; femoral vascular conductance (FVC) was computed (FBF/ABP). Responses evoked by 5-minute periods of hypoxia (breathing 8% O₂) and adenosine infusion (1.2mg.kg^-1.hr^-1 ia) were recorded before and after, the Nox inhibitor apocynin (13mg.kg^-1 iv, Group 1), the NO synthase inhibitor L-NAME (10mg.kg^-1 iv, so as to maximize ROS levels) followed by apocynin (Group 2) and L-NAME followed by a second Nox inhibitor DPI (Group 3). The muscle vasodilatation was analysed as the change in the integral of FVC (IntFVC) in arbitrary conductance units (CU). Comparisons were made by ANOVA, P<0.05 being considered significant. Acute hypoxia induced a fall in ABP (from 103±3 to 50±4mmHg), and muscle vasodilatation (IntFVC increased from 2.8±0.4CU by 2.6±0.4CU). In Group 1, apocynin had no effect on the hypoxia-induced increase in IntFVC. As expected, in Group 2, L-NAME increased baseline ABP (to 130±1mmHg) and decreased IntFVC (to 1.6±0.2CU) reflecting removal of the effect of tonic NO release, and the hypoxia-induced increase in IntFVC was attenuated (1.2±0.2CU), but apocynin had no effect on baseline (1.7±0.2CU) or on the hypoxia-induced dilatation (1.4±0.2CU). Similarly, in Group 3, DPI had no effect on the hypoxia-induced increase in IntFVC after L-NAME. Moreover, adenosine infusion induced similar dilator responses to hypoxia, but they were not affected by apocynin or DPI. The pharmacological inhibitors of Nox currently available are not fully selective and when used individually, may have led to overestimation of the role of Nox. The lack of effect of apocynin or DPI in the present study leads us to propose that Nox-derived ROS do not modulate the vasodilatation induced in skeletal muscle by acute systemic hypoxia, even when the inhibition of NO synthase prevents the scavenging of ROS by NO. It may be that a chronic condition, such as chronic hypoxia, is required increase Nox activity in systemic tissues.