—Transgenic mouse models have been developed to manipulate β-adrenergic receptor (βAR) signal transduction. Although several of these models have altered βAR subtypes, the specific functional sequelae of βAR stimulation in murine heart, particularly those of β₂-adrenergic receptor (β₂AR) stimulation, have not been characterized. In the present study, we investigated effects of β₂AR stimulation on contraction, [Ca²⁺]_i transient, and L-type Ca²⁺ currents (I_Ca) in single ventricular myocytes isolated from transgenic mice overexpressing human β₂AR (TG4 mice) and wild-type (WT) littermates. Baseline contractility of TG4 heart cells was increased by 3-fold relative to WT controls as a result of the presence of spontaneous β₂AR activation. In contrast, β₂AR stimulation by zinterol or isoproterenol plus a selective β₁-adrenergic receptor (β₁AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, β₂AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the I_Ca, [Ca²⁺]_i, and contractile responses to β₂AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac β₂AR response is mediated by cAMP-dependent mechanisms, because it was totally blocked by the inhibitory cAMP analog Rp-cAMPS. These results suggest that PTX-sensitive G proteins are responsible for the unresponsiveness of mouse heart to agonist-induced β₂AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [γ-³²P]GTP azidoanilide into α subunits of G_i2 and G_i3 after β₂AR stimulation by zinterol or isoproterenol plus the β₁AR blocker CGP 20712A. This effect to activate G_i proteins was abolished by a selective β₂AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) β₂ARs in murine cardiac myocytes couple to concurrent G_s and G_i signaling, resulting in null inotropic response, unless the G_i signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to β₂AR agonists in TG4 mice is not due to a saturation of cell contractility or of the cAMP signaling cascade but rather to an activation of β₂AR-coupled G_i proteins; and (3) spontaneous β₂AR activation may differ from agonist-stimulated β₂AR signaling.