Nitrate tolerance is associated with an enhanced superoxide anion production and can be attenuated by statins, which interact with the 2 main [eNOS and NAD(P)H oxidase] pathways involved in producing this oxidative stress. Three groups of normocholesterolemic rats were treated: group 1 received rosuvastatin (10 mg/kg/d PO) for 5 weeks and in the last 3 days cotreatment with nitroglycerin (NTG 50 mg/kg/d, subcutaneous injections BID); group 2 received only NTG (50 mg/kg/d BID for the last 3 days); and group 3 served as control. Rings of thoracic aortas from these groups were studied in organ baths. Relaxations to NTG (0.1 nM to 0.1 mM) were determined on phenylephrine-preconstricted rings and O2 production (RLU/10 s/mg dry weight) was assessed by lucigenin and the luminol analogue (L-012) chemiluminescence technique. In group 2 (NTG), the concentration-response curves to NTG were significantly shifted to the right: the pD2 (-log NTG concentration evoking a half-maximal relaxation) was 6.75+/-0.06 (n=7) versus 7.75+/-0.07 (n=7) in group 3 (not exposed to NTG, P<0.05); O2 production was enhanced (10,060+/-1,205, n=7 versus 5,235+/-1,052, n=7; P<0.05). In contrast, in group 1, the rightward shift was attenuated: pD2 value was 7.20+/-0.10 (n=8), P<0.05 versus group 2; O2 production was decreased (5911+/-663; n=9, P<0.05 versus group 2). In addition, before NTG exposure, rosuvastatin treatment decreased p22phox [the essential NAD(P)H oxidase subunit] abundance in the aortic wall and decreased NAD(P)H oxidase activity. In contrast, this treatment did not alter either eNOS abundance or the basal release of endothelium-derived NO. Interestingly, in vivo treatment with apocynin, an NAD(P)H oxidase inhibitor, produced a protection similar to that with rosuvastatin. Long-term rosuvastatin treatment protects against nitrate tolerance in the rat aorta by counteracting NTG-induced increase in O2 production. This protection seems to involve a direct interaction with the NAD(P)H oxidase pathway rather than an up-regulation of the eNOS pathway.