T he 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 hyper-responsiveness 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.