The α subunit of the stimulatory heterotrimeric G protein (G_sα) is critical for the β-adrenergic receptor activation of the cAMP messenger system. The role of G_sα in regulating cardiac Ca²⁺ channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac G_sα were used to assess the role of G_sα on the whole-cell Ca²⁺ currents (I_Ca). Cardiac myocytes from transgenic mice had a 490% higher peak I_Ca compared with those of either wild-type controls or G_sα-nonexpressing littermates. The effect of G_sα overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPγS-activated G_sα. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated G_sα. The data indicate that G_sα overexpression is associated with a constitutive enhancement of I_Ca which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.