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We investigated the contribution of the intracellular calcium (Ca) 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 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 transient, like the hyperpolarization-activated “funny current” ( If) and the ACh-sensitive potassium current ( IK,ACh), is an important determinant of ACh-mediated pacemaker slowing. When If and IK,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 If and IK,ACh-inhibited SAN cells, good correlations were found between the ACh-mediated change in interbeat interval and the ACh-mediated change in Ca transient decay ( r2 = 0.98) and slow diastolic Ca rise ( r2 = 0.73). Inhibition of the Ca transient by ryanodine (3 μM) or BAPTA-AM (5 μM) facilitated ACh-mediated pacemaker slowing. Furthermore, ACh depressed the Ca transient and reduced the sarcoplasmic reticulum (SR) Ca2+ content, all in a concentration-dependent fashion. At 1 μM ACh, the spontaneous activity and Ca transient were abolished, but completely recovered when cAMP production was stimulated by forskolin (10 μM) and IK,ACh was inhibited by tertiapin (100 nM). Also, inhibition of the Ca transient by ryanodine (3 μM) or BAPTA-AM (25 μM) exaggerated the ACh-mediated inhibition of cAMP content, indicating that Ca affects cAMP production in SAN cells. In conclusion, muscarinic receptor stimulation inhibits the Ca transient via a cAMP-dependent signaling pathway. Inhibition of the Ca transient contributes to pacemaker slowing and inhibits Ca-stimulated cAMP production. Thus, we provide functional evidence for the contribution of the Ca transient to ACh-induced inhibition of pacemaker activity and cAMP content in rabbit SAN cells. [ABSTRACT FROM AUTHOR]