Background and aim: We have demonstrated that overactivation of β-adrenoceptors induces oxidative stress and increases vasoconstrictor responses in rat aorta. This study focusing to investigate the β-adrenoceptor subtype as well the signaling pathway involved in these vascular effects. Methods and results: Male, 3-mo-old mice lacking β1- or β2-adrenoceptors subtypes (β1KO, β2KO, N=12-16) and wild-type (WT, N=14) were daily treated with isoproterenol (ISO, 15 mg/kg/day, s.c.) or vehicle for 7 days. ISO treatment significantly (p<0.05, One-Way ANOVA) enhanced the vasoconstrictor response to phenylephrine (PHE) in aorta from WT and β1KO mice as compared to vehicle (34 and 35% of increase in Emax to PHE, respectively), but not in β2KO. Nitric oxide synthase inhibitor L-NAME (100 μM) increased Emax to PHE in aorta from all groups evaluated. This effect was lesser pronounced in aorta from ISO-treated WT and β1KO mice than vehicle-treated groups, but was similar between ISO-treated and non-treated β2KO mice. Incubation with superoxide dismutase (SOD, 150 U/mL) normalized increased Emax to PHE observed in aortic rings from ISO-treated WT and β1KO mice, but did not significantly change this response in ISO-treated β2KO mice. Immunoblotting experiments revealed increased expression of Giα protein (+50%) and of phosphorylated[Thr202/Tyr204]-ERK1/2 (+90%) in aorta from ISO-treated WT compared to vehicle WT. In addition, incubation with Gi inhibitor pertussis toxin (PTx, 4 μM) normalized the enhanced Emax to PHE observed in aortic rings from ISO-treated WT mice. ISO treatment enhanced fluorescence to hydroethidine (+100%) in aortas from WT, indicating oxidative stress that was significantly reduced by SOD and PTx. These effects of ISO were abolished in β2KO mice. Conclusions: Giα protein coupled to β2-adrenoceptor plays a pivotal role in the oxidative stress and endothelial dysfunction induced by persistent β-adrenergic activation.