Cardiac automaticity is controlled by G protein-coupled receptors, such as adrenergic, muscarinic, and adenosine receptors. The strength and duration of G protein signaling is attenuated by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Galpha subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in Galpha subunits that disrupt Galpha-RGS binding and introduced them into embryonic stem (ES) cells by homologous recombination. Spontaneously contacting cardiocytes derived from the ES cells were used to evaluate the role of endogenous RGS proteins in chronotropic regulation. The RGS-insensitive GalphaoG184S homozygous knock-in (GalphaoGS/GS) cells demonstrated enhanced adenosine A1 and muscarinic M2 receptor-mediated bradycardic responses. In contrast, Galphai2GS/GS cells showed enhanced responses to M2 but not A1 receptors. Similarly M2 but not A1 bradycardic responses were dramatically enhanced in Galphai2GS/GS mice. Blocking G protein-coupled inward rectifying K+ (GIRK) channels largely abolished the mutation-induced enhancement of the M2 receptor-mediated response but had a minimal effect on A1 responses. The Galphas-dependent stimulation of beating rate by the beta2 adrenergic receptor agonist procaterol was significantly attenuated in GalphaoGS/GS and nearly abolished in Galphai2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Galpha(i/o)-linked receptors on cardiac automaticity. Furthermore, M2 and A1 receptors differentially use Galphai2 and Galphao and associated downstream effectors. Thus, alterations in RGS function may play a role in pathophysiological processes and RGS proteins could represent novel cardiovascular therapeutic targets.