The purpose of the present study was to examine the role of Gi2α in Ca2+channel regulation using Gi2α gene knockout mouse ventricular myocytes. The whole cell voltage-clamp technique was used to study the effects of the muscarinic agonist carbachol (CCh) and the β-adrenergic agonist isoproterenol (Iso) on cardiac L-type Ca2+currents in both 129Sv wild-type (WT) and Gi2α gene knockout (Gi2α−/−) mice. Perfusion with CCh significantly inhibited the Ca2+current in WT cells, and this effect was reversed by adding atropine to the CCh-containing solution. In contrast, CCh did not affect Ca2+currents in Gi2α−/− ventricular myocytes. Addition of CCh to Iso-containing solutions attenuated the Iso-stimulated Ca2+current in WT cardiomyocytes but not in Gi2α−/− cells. These findings demonstrate that, whereas the Iso-Gsα signal pathway is intact in Gi2α gene knockout mouse hearts, these cells lack the inhibitory regulation of Ca2+channels by CCh. Therefore, Gi2α is necessary for the muscarinic regulation of Ca2+channels in the mouse heart. Further studies are needed to delineate the possible interaction of Giand other cell signaling proteins and to clarify the level of interaction of G protein-coupled regulation of L-type Ca2+current in the heart.