Background —Nebivolol is a β ₁ -selective adrenergic receptor antagonist with proposed nitric oxide (NO)–mediated vasodilating properties in humans. In this study, we explored whether nebivolol indeed induces NO production and, if so, by what mechanism. We hypothesized that not nebivolol itself but rather its metabolites augment NO production. Methods and Results —Mouse thoracic aorta segments were bathed in an organ chamber. Administration of nebivolol did not affect NO production. When nebivolol was allowed to metabolize in vivo in mice, addition of plasma of these mice caused a sustained 2-fold increase in NO release. Interestingly, coadministration of a selective β ₂ -adrenergic receptor antagonist (butoxamine) prevented the response. Immunohistochemistry and Western blot analysis demonstrated the presence of β ₂ - but not β ₁ -adrenergic receptors on endothelial cells. In the absence of calcium, metabolized nebivolol failed to increase NO production, suggesting a role for calcium-dependent NO synthase. With digital fluorescence imaging, a rapid and sustained rise in endothelial cytosolic free Ca ²⁺ concentration was observed after administration of metabolized nebivolol, which also was abrogated by butoxamine pretreatment. Conclusions —In vivo metabolized nebivolol increases vascular NO production. This phenomenon involves endothelial β ₂ -adrenergic receptor ligation, with a subsequent rise in endothelial free [Ca ²⁺ ] _i and endothelial NO synthase–dependent NO production. This may be an important mechanism underlying the nebivolol-induced, NO-mediated arterial dilation in humans.