Ageing has been proposed to be associated with increased levels of reactive oxygen species (ROS) that scavenge nitric oxide (NO). In eight young sedentary (23±1 years; Y), eight older lifelong sedentary (66±2 years; OS) and eight older lifelong physically active subjects (62±2 years; OA), we studied the effect of ROS on systemic and skeletal muscle NO bioavailability and leg blood flow by infusion of the antioxidant N-acetylcysteine (NAC). Infusion of NAC increased the bioavailability of NO in OS, as evidenced by an increased concentration of stable metabolites of NO (NOx) in the arterial and venous circulation and in the muscle interstitium. In OA, infusion of NAC only increased NOx concentrations in venous plasma whereas in Y, infusion of NAC did not affect NOx concentrations. Skeletal muscle protein levels of endothelial and neuronal NO synthase were 32% and 24% higher, respectively, in OA than in OS. Exercise at 12 W elicited a lower leg blood flow response that was associated with a lower leg oxygen uptake in OS than in Y. The improved bioavailability of NO in OS did not increase blood flow during exercise. These data demonstrate that NO bioavailability is compromised in the systemic circulation and in the musculature of sedentary ageing humans due to increased oxidative stress. Lifelong physical activity opposes this effect within the trained musculature and in the arterial circulation. The lower blood flow response to leg exercise in ageing humans is not associated with a reduced NO bioavailability.