Objective: Sustained β-adrenergic stimulation is a hallmark of sympathetic hyperactivity in cardiovascular diseases. It is associated with oxidative stress and altered vasoconstrictor tone. This study investigated the β-adrenoceptor subtype and the signaling pathways implicated in the vascular effects of β-adrenoceptor overactivation. Methods and Results: Mice lacking the β1- or β2-adrenoceptor subtype (β1KO, β2KO) and wild-type (WT) were treated with isoproterenol (ISO, 15 μg.g-1.day-1, 7 days). ISO significantly enhanced the maximal vasoconstrictor response (Emax) of the aorta to phenylephrine in WT ( +34%) and β1KO mice (+35%) but not in β2KO mice. The nitric oxide synthase (NOS) inhibitor L-NAME abolished the differences in phenylephrine response between the groups, suggesting that ISO impaired basal NO availability in the aorta of WT and β1KO mice. Superoxide dismutase (SOD), pertussis toxin (PTx) or PD 98,059 (p-ERK 1/2 inhibitor) incubation reversed the hypercontractility of aortic rings from ISO-treated WT mice; aortic contraction of ISO-treated β2KO mice was not altered. Immunoblotting revealed increased aortic expression of Giα-3 protein (+50%) and phosphorylated ERK1/2 (+90%) and decreased eNOS dimer/monomer ratio in ISO-treated WT mice. ISO enhanced the fluorescence response to dihydroethidium (+100%) in aortas from WT mice, indicating oxidative stress that was normalized by SOD, PTx and L-NAME. The ISO effects were abolished in β2KO mice. Conclusions: The β2-adrenoceptor/Giα signaling pathway is implicated in the enhanced vasoconstrictor response and eNOS uncoupling-mediated oxidative stress due to ISO treatment. 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