Parasympathetic stimulation of the heart acts through M2-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins G&agr;i/o, the specific signal transduction requirements of G&agr;i2and G&agr;i3in mediating muscarinic regulated L-type calcium currents (ICa, L), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from G&agr;i2-null mice, G&agr;i3-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, andICa, Lwere all measured in response to isoproterenol, a &bgr;-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and G&agr;i3-null cells but had no effect in G&agr;i2-null cells. This study demonstrates that G&agr;i2, but not G&agr;i3, is required for muscarinic inhibition of the &bgr;-adrenergic response in adult murine ventricular myocytes.