G protein-coupled receptors play a pivotal role in regulating cardiac automaticity. Their function is controlled by regulator of G protein signaling (RGS) proteins acting as GTPase-activating proteins for Gα subunits to suppress Gαiand Gαqsignaling. Using knock-in mice in which Gαi2-RGS binding and negative regulation are disrupted by a genomic Gαi2G184S (GS) point mutation, we recently (Fu Y, Huang X, Zhong H, Mortensen RM, D'Alecy LG, Neubig RR. Circ Res 98: 659–666, 2006) showed that endogenous RGS proteins suppress muscarinic receptor-mediated bradycardia. To determine whether this was due to direct regulation of cardiac pacemakers or to alterations in the central nervous system or vascular responses, we examined isolated, perfused hearts. Isoproterenol-stimulated beating rates of heterozygote (+/GS) and homozygote (GS/GS) hearts were significantly more sensitive to inhibition by carbachol than were those of wild type (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced fivefold in GS/GS and twofold in +/GS hearts compared with +/+. A1-adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the sinoatrial node, +/GS and GS/GS hearts show significantly increased carbachol-induced third-degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared with +/+. Thus loss of the inhibitory action of endogenous RGS proteins on Gαi2potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in Gαi2G184S mice suggests a possible role for alterations of Gαi2or RGS proteins in sick sinus syndrome and pathological AV block.