We investigated the contribution of the intracellular calcium (Ca i 2+ ) transient to acetylcholine (ACh)-mediated reduction of pacemaker frequency and cAMP content in rabbit sinoatrial nodal (SAN) cells. Action potentials (whole cell perforated patch clamp) and Ca i 2+ transients (Indo-1 fluorescence) were recorded from single isolated rabbit SAN cells, whereas intracellular cAMP content was measured in SAN cell suspensions using a cAMP assay (LANCE®). Our data show that the Ca i 2+ transient, like the hyperpolarization-activated “funny current” (I f) and the ACh-sensitive potassium current (I K,ACh), is an important determinant of ACh-mediated pacemaker slowing. When I f and I K,ACh were both inhibited, by cesium (2 mM) and tertiapin (100 nM), respectively, 1 μM ACh was still able to reduce pacemaker frequency by 72%. In these I f and I K,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca i 2+ transient decay (r 2 = 0.98) and slow diastolic Ca i 2+ rise (r 2 = 0.73). Inhibition of the Ca i 2+ transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca i 2+ transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Ca i 2+ transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and I K,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Ca i 2+ transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca i 2+ affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca i 2+ transient via a cAMP-dependent signaling pathway. Inhibition of the Ca i 2+ transient contributes to pacemaker slowing and inhibits Ca i 2+ -stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca i 2+ transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells.