Transgenic mouse models have been developed to manipulate beta-adrenergic receptor (beta AR) signal transduction. Although several of these models have altered beta AR subtypes, the specific functional sequelae of beta AR stimulation in murine heart, particularly those of beta2-adrenergicreceptor (beta2AR) stimulation, have not been characterized. In the present study, we investigated effects of beta (2) AR stimulation on contraction, [Ca2+]itransient, and L-type Ca2+currents (ICa) in single ventricular myocytes isolated from transgenic mice overexpressing human beta2AR (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 beta2AR activation. In contrast, beta2AR stimulation by zinterol or isoproterenol plus a selective beta1-adrenergicreceptor (beta1AR) antagonist CGP 20712A failed to enhance the contractility in TG4 myocytes, and more surprisingly, beta2AR stimulation was also ineffective in increasing contractility in WT myocytes. Pertussis toxin (PTX) treatment fully rescued the ICa, [Ca2+]i, and contractile responses to beta2AR agonists in both WT and TG4 cells. The PTX-rescued murine cardiac beta2AR 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 beta2AR stimulation. This was further corroborated by an increased incorporation of the photoreactive GTP analog [gamma-(32) P]GTP azidoanilide into alpha subunits of Gi2and Gi3after beta2AR stimulation by zinterol or isoproterenol plus the beta1AR blocker CGP 20712A. This effect to activate Giproteins was abolished by a selective beta2AR blocker ICI 118,551 or by PTX treatment. Thus, we conclude that (1) beta2ARs in murine cardiac myocytes couple to concurrent Gsand Gisignaling, resulting in null inotropic response, unless the G (i) signaling is inhibited; (2) as a special case, the lack of cardiac contractile response to beta2AR 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 beta (2) AR-coupled Giproteins; and (3) spontaneous beta2AR activation may differ from agonist-stimulated beta2AR signaling. (Circ Res. 1999;84:43-52.)