In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gsand Gi, the functional role of Gois still poorly defined. To investigate the role of Gαoin the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of Gαo1* (Gαo*), the predominant Gαoisoform in the heart. Gαoexpression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in Gαo* transgenic mice in vivo, along with increased L-type Ca2+channel current density, calcium transients, and cell shortening in ventricular Gαo*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in Gαo* ventricles. Density and affinity of β-adrenoceptors, cAMP levels, and PKA activity were comparable in Gαo* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon Gαo* expression, particularly in the vicinity of the ryanodine receptor. We conclude that Gαo* exerts a positive effect on Ca2+cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+handling proteins in hearts with constitutive Gαoactivation.