Recent studies have indicated that Ca²⁺ cycling by the sarcoplasmic reticulum (referred to as a Ca²⁺-clock) within sinoatrial node cells (SANC) generates rhythmic, spontaneous local Ca²⁺ releases (LCR's) that are adenylyl cyclase (AC)-cAMP-protein kinase A (PKA)-dependent. The purpose of the present study was to determine how cholinergic receptor (ChR) signaling effects, initiated by carbachol (CCh), on AC, cAMP, PKA, sarcolemmal ion channels, and LCRs become integrated to cause beating rate reduction (BRR) in single, isolated rabbit SANC. The threshold [CCh] for BRR was ~10 nM; half maximal inhibition (IC₅₀) was achieved at 100 nM; and 1000 nM stopped spontaneous beating. G_i inhibition by pertussis toxin blocked all CCh effects on BRR. Blockade of I_f did not affect BRR at any [CCh]. I_KACh activation, evidenced by hyperpolarization, became apparent at [CCh]>30 nM. At IC₅₀, CCh reduced cAMP, and reduced PKA-dependent phospholamban (PLB) phosphorylation by ~50%. When I_KACh was blocked (tertiapin Q, 1 µM) the dose response of BRR to CCh mirrored that of CCh to dephosphorylate PLB. At IC₅₀, CCh caused a time-dependent reduction in the LCR number and size and a time-dependent increase in LCR period that paralleled coincident BRR. The phosphatase inhibitor, calyculin A, reversed the effect of IC₅₀ CCh on both LCR's and BRR. Numerical model simulations provided theoretical support for the idea that LCR-induced activation of NCX current is integrated into the cholinergic modulation of BRR. Thus, ChR-induced BRR requires G_i activation, and the extent to which G_i couples to Ca²⁺ cycling via PKA signaling, or to I_KACh: at low [CCh] BRR is attributable to a suppression of PKA-dependent Ca²⁺ signaling; as [CCh] increases beyond 30 nM, a tight coupling between suppression of PKA-dependent Ca²⁺ signaling and I_KACh activation underlies a more pronounced BRR.