Two subtypes of β-adrenoceptors, β₁ and β₂, mediate cardiac catecholamine effects. These two types differ qualitatively, e.g., regarding G protein coupling and calcium channel stimulation. Transgenic mice overexpressing human β₂-adrenoceptors survive high-expression levels, unlike mice overexpressing β₁-adrenoceptors. We examined the role of inhibitory G_i proteins, known to be activated by β₂- but not β₁-adrenoceptors, on the chronic effects of human β₂-adrenoreceptor overexpression in transgenic mice. These mice were crossbred with mice where Gα_i2, a functionally important cardiac G_i α-subunit, was inactivated by targeted gene deletion. Survival of β₂-adrenoreceptor transgenic mice was reduced by heterozygous inactivation of Gα_i2. Homozygous knockout/β₂-adrenoreceptor transgenic mice died within 4 days after birth. Heterozygous knockout/β₂-adrenoreceptor transgenic mice developed more pronounced cardiac hypertrophy and earlier heart failure compared with β₂-adrenoreceptor transgenic mice. Single calcium-channel activity was strongly suppressed in heterozygous knockout/β₂-adrenoreceptor transgenic mice. In cardiomyocytes from these mice, pertussis toxin treatment in vitro fully restored channel activity and enhanced channel activity in cells from homozygous Gα_i2 knockout animals. Cardiac Gα_i3 protein was increased in all Gα_i2 knockout mouse strains. Our results demonstrate that Gα_i2 takes an essential protective part in chronic signaling of overexpressed β₂-adrenoceptors, leading to prolonged survival and delayed cardiac pathology. However, reduction of calcium-channel activity by β₂-adrenoreceptor overexpression is due to a different pertussis-toxin-sensitive pathway, most likely by Gα_i3. This result indicates that subtype-specific signaling of β₂-adrenoreceptor functionally bifurcates at the level of G_i, leading to different effects depending on the Gα isoform.