Ca²⁺ influx through voltage-gated R-type (Ca_V2.3) Ca²⁺ channels is important for hormone and neurotransmitter secretion and other cellular events. Previous studies have shown that Ca_V2.3 is both inhibited and stimulated through signaling mechanisms coupled to muscarinic acetylcholine receptors. We previously demonstrated that muscarinic stimulation of Ca_V2.3 is blocked by regulator of G protein signaling 2 (RGS2). Here, we investigated whether muscarinic inhibition of Ca_V2.3 is antagonized by RGS3. RGS3 is particularly interesting because it contains a lengthy (~380 residue) amino-terminal domain of uncertain physiological function. Ca_V2.3, M₂ muscarinic acetylcholine receptors (M₂R) and various deletion mutants of RGS3, including its native isoform RGS3T, were expressed in HEK293 cells, and agonist-dependent inhibition of Ca_V2.3 was quantified using whole-cell patch-clamp recordings. Full-length RGS3, RGS3T and the core domain of RGS3 were equally effective in antagonizing inhibition of Ca_V2.3 through M₂R. These results identify RGS3 and RGS3T as potential physiological regulators of R-type Ca²⁺ channels. Furthermore, they suggest that the signaling activity of RGS3 is unaffected by its extended amino-terminal domain. Confocal microscopy was used to examine the intracellular locations of four RGS3-EGFP fusion proteins. The RGS3 core domain was uniformly distributed throughout both cytoplasm and nucleus. By contrast, full-length RGS3, RGS3T and the amino-terminal domain of RGS3 were restricted to the cytoplasm. These observations suggest that the amino-terminus of RGS3 may serve to confine it to the cytoplasmic compartment where it can interact with cell surface receptors, heterotrimeric G proteins and other signaling proteins.