The development of nitrate tolerance has been found to be associated with vascular production of superoxide anion (O2-*), generated mainly by the eNOS and NADPH oxidase pathways. The aim of our study was to investigate whether long-term angiotensin-converting enzyme inhibition by ramipril is able to protect against nitrate tolerance in the aortas of eNOS-deficient (eNOS-/-) mice and to assess the implication of the NADPH oxidase pathway. Therefore, 3 types of treatment were given to wild-type (WT) and eNOS-/- mice: group 1 received ramipril for 5 weeks and a co-treatment with ramirpil plus nitroglycerine (NTG) during the last 4 days, group 2 received only NTG, and group 3 served as control. Relaxations to NTG (0.1 nmol/L to 0.1 mmol/L) were determined on U44619, a thromboxane analogue, precontracted rings, and O2-* production were assessed on aorta homogenates with the lucigenin-enhanced chemiluminescence technique. Cyclic guanosine monophosphate and reverse-transcriptase-polymerase chain reaction analyses were performed on whole mouse aortas. In WT group 2, the concentration-effect curves to NTG were significantly shifted to the right: the pD2 was 6.16 +/- 0.17 (n = 6) vs 6.81 +/- 0.10 (n = 6) in WT group 3 (not exposed to NTG; P < 0.05) and O2-* production was enhanced from 100% +/- 11% (n = 9) to 191% +/- 21% (n = 6; P < 0.01). In contrast, in WT group 1, the rightward shift was abolished: the pD2 value was 6.73 +/- 0.13 (n = 6; NS vs group 3 WT) and O2-* production was 117% +/- 6% (n = 7; NS vs group 3 WT). In eNOS groups 1 and 3, similar data were observed: the pD2 values were 7.58 +/- 0.08 and 7.38 +/- 0.11 (NS) vs 6.89 +/- 0.20 in eNOS group 2 (n = 6; P < 0.01). In the WT mice aortas, ramipril treatment significantly increased the cyclic guanosine monophosphate levels (reflecting nitric oxide availability), which returned to control values after in vivo co-treatment with a bradykinin BK2 antagonist (Icatibant). In both strains, candesartan, an AT1 blocker, was also able to protect against the development of nitrate tolerance. Moreover, before NTG exposure, ramipril treatment decreased p22phox and gp91phox (essential NADPH oxidase subunits) mRNA expression in aortas from both mice strains. In conclusion, long-term ramipril treatment in mice protects against the development of nitrate tolerance by counteracting NTG-induced increase in O2 production, which involves a direct interaction with the NADPH oxidase pathway and seems to be completely independent of the eNOS pathway.