The cardiac β-adrenergic pathway potently stimulates myocardial performance, thereby providing a mechanism for myocardial contractile reserve. β-Adrenergic activation also increases cardiac nitric oxide (NO) production, which attenuates positive inotropy, suggesting a possible negative feedback mechanism. Recently, in vitro studies suggest that stimulation of the β3-adrenoceptor results in a negative inotropic effect through NO signaling. In this study, using mice with homozygous β3-adrenoceptor deletion mutations, we tested the hypothesis that the β3-adrenoceptor is responsible for β-adrenergic activation of NO. Although resting indices of myocardial contraction were similar, β-adrenergic-stimulated inotropy was increased in β3(-/-) mice, and similar hyperresponsiveness was seen in mice lacking endothelial NO synthase (NOS3). NOS inhibition augmented isoproterenol-stimulated inotropy in wild-type (WT), but not in β3(-/-) mice. Moreover, isoproterenol increased myocardial cGMP in WT, but not β3(-/-), mice. NOS3 protein abundance was not changed in β3(-/-) mice, and cardiac β3-adrenoceptor mRNA was detected in both NOS3(-/-) and WT mice. These findings indicate that the β3-adrenergic subtype participates in NO-mediated negative feedback over β-adrenergic stimulation.