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 (GAPs) for G subunits to suppress G_i and G_q signaling. Using knock-in mice in which G_i2-RGS binding and negative regulation is disrupted by a genomic G_i2G184S (GS) point mutation, we recently (Fu et al Circ Res 98:659-66, 2006) showed that endogenous RGS proteins suppress muscarinic receptor (M₂R)-mediated bradycardia. To determine if 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 wildtype (+/+). Even greater effects were seen in the absence of isoproterenol; the potency of muscarinic-mediated bradycardia was enhanced 5-fold in GS/GS and 2-fold in +/GS hearts compared to +/+. A₁ adenosine receptor-mediated bradycardia was unaffected. In addition to effects on the SA node, +/GS and GS/GS hearts show significantly increased carbachol-induced 3^rd degree atrioventricular (AV) block. Atrial pacing studies demonstrated an increased PR interval and AV effective refractory period in GS/GS hearts compared to +/+. Thus, loss of the inhibitory action of endogenous RGS proteins on G_i2 potentiates muscarinic inhibition of cardiac automaticity and conduction. The severe carbachol-induced sinus bradycardia in G_i2G184S mice suggests a possible role for alterations of G_i2 or RGS proteins in sick sinus syndrome and pathologic AV block.