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 Gα subunits; however, little is known about the role of endogenous RGS proteins in cardiac function. We created point mutations in Gα subunits that disrupt Gα-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 GαoG184S homozygous knock-in (GαoGS/GS) cells demonstrated enhanced adenosine A1and muscarinic M2receptor–mediated bradycardic responses. In contrast, Gαi2GS/GS cells showed enhanced responses to M2but not A1receptors. Similarly M2but not A1bradycardic responses were dramatically enhanced in Gαi2GS/GS mice. Blocking G protein–coupled inward rectifying K+(GIRK) channels largely abolished the mutation-induced enhancement of the M2receptor–mediated response but had a minimal effect on A1responses. The Gαs-dependent stimulation of beating rate by the β2adrenergic receptor agonist procaterol was significantly attenuated in GαoGS/GS and nearly abolished in Gαi2GS/GS cells because of enhanced signaling via a pertussis toxin sensitive mechanism. Thus, endogenous RGS proteins potently reduce the actions of Gαi/o-linked receptors on cardiac automaticity. Furthermore, M2and A1receptors differentially use Gαi2and Gαoand associated downstream effectors. Thus, alterations in RGS function may play a role in pathophysiological processes and RGS proteins could represent novel cardiovascular therapeutic targets.