Overactivation of β-adrenoceptors induced by isoproterenol (ISO) treatment causes cardiac remodeling and vascular dysfunction. It is known that angiotensin II via AT1 receptor and aldosterone via mineralocorticoid receptor (MR) partially mediates the cardiac injury induced by ISO, including: myocardial hypertrophy and fibrosis, oxidative stress and inflammation. In the present study we tested the hypothesis that signaling pathways activated by AT1 and/or MR receptors are involved in the impairment of nitric oxide (NO) bioavailability induced by ISO treatment. Male Wistar rats were treated with ISO (0.3 mg/kg/day, s.c.) or vehicle (CT) and co-treated or not with the AT1 antagonist losartan (LOS, 40 mg/kg/day, p.o.) or MR antagonist spironolactone (SPI, 200 mg/kg/day, p.o.) for one week. In isolated aortic rings with endothelium (n=6-8 in each group), maximal response (Rmax) evoked by phenylephrine (Phe, 1ηM- 10μM) was enhanced in ISO-treated rats (CT= 6.8 ± 0.4 vs. ISO= 8.8 ± 0.5 mN/mm; p<0.05; n=6-8). LOS co-treatment did not modify this contractile hyperresponsiveness (ISO/LOS= 8.3 ± 0.2 mN/mm; n=6; p<0.05 vs. ISO), while SPI abolished this effect (ISO/SPI= 6.3 ± 0.3 mN/mm; n=8; p<0.05 vs. ISO). The NO synthase (NOS) inhibitor L-NAME (100 μM) enhanced the Rmax to Phe in aortas from all groups, abolishing differences among then. The superoxide dismutase (SOD, 150 U/mL) reduced Rmax to Phe only in ISO and ISO/LOS groups. This result suggests an enhanced participation of superoxide anion on aortic contractile response of ISO-treated rats that is normalized by SPI but not altered by LOS. Fluorescences to hydroethidine (DHE, 2 μM) and to diaminofluorescein-2 (DAF-2, 8 μM) were used to quantify reactive oxygen species and NO, respectively (n=5-6 in each group). Elevated DHE (+44%) and reduced DAF-2 (-60%) fluorescence was observed in aortas from ISO-treated rats; SPI co-treatment, but not LOS, normalized it. Incubation with L-NAME significantly reduced the fluorescence to DHE (-33%) in aortic slices of ISO group. Protein expression was evaluated in aortic homogenates (n=6-8 in each group) and no significant changes in SOD-1, -2 and -3 protein expression among groups were observed. On the other hand, eNOS protein expression was significantly enhanced by ISO treatment (+56%) whilst phosphorylation of eNOS at Ser1177 (normalized by total eNOS content) was reduced (-43%). Neither LOS nor SPI modified this ISO effect. In conclusion, data suggest that ISO treatment could enhance vascular contractility due to high superoxide anion production via uncoupled eNOS and reduced NO availability. These vascular effects seem to be mediated by MR but not by AT1 receptor.