Muscarinic acetylcholine receptors (mAChR) are known to mediate the acetylcholine inhibition of Ca²⁺ channels in central and peripheral neurons. Stellate ganglion (SG) neurons provide the main sympathetic input to the heart and contribute to the regulation of heart rate and myocardial contractility. Little information is available regarding mAChR regulation of Ca²⁺ channels in SG neurons. The purpose of the present study was to identify the mAChR subtypes that modulate Ca²⁺ channel currents in rat SG neurons innervating heart muscle. Accordingly, the modulation of Ca²⁺ channel currents by the muscarinic cholinergic agonist, oxotremorine-methiodide (Oxo-M) and mAChR blockers was examined. Oxo-M-mediated mAChR stimulation led to inhibition of Ca²⁺ currents through voltage-dependent (VD) and voltage-independent (VI) pathways. Preexposure of SG neurons to the M₁ receptor blocker, M₁-toxin, resulted in VD inhibition of Ca²⁺ currents following Oxo-M application. On the other hand, VI modulation of Ca²⁺ currents was observed following pretreatment of cells with methoctramine (M₂ mAChR blocker). The Oxo-M-mediated inhibition was nearly eliminated in the presence of both M₁ and M₂ mAChR blockers, but was unaltered when SG neurons were exposed to the M₄ mAChR toxin, M₄-toxin. Finally, the results from single-cell RT-PCR and immunofluorescence assays indicated that M₁ and M₂ receptors are expressed and located on the surface of SG neurons. Overall, the results indicate that SG neurons which innervate cardiac muscle express M₁ and M₂ mAChR, and activation of these receptors leads to inhibition of Ca²⁺ channel currents through VI and VD pathways, respectively.