Proposed mechanisms by which α2-adrenergic receptors (α2AR) regulate intracellular calcium ([Ca2+](i)) include stimulation and inhibition of cell surface calcium channels, stimulation of calcium release via receptor coupling to G(q) with subsequent activation of phospholipase C and release of IP3, or stimulation of calcium release via coupling to G(i) in an IP3- independent manner. These potential mechanisms were explored in cells that expressed α(2A)R endogenously (HEL cells), permanently transfected CHO cells, and transiently transfected COS-7 cells. Each cell type displayed agonist (UK14304)-dependent increases in [Ca2+](i) that were blocked by yohimbine, ablated by pertussis toxin, and largely unaffected by chelation of extracellular calcium. Furthermore, calcium release was associated with IP3 accumulation and was blocked by an inhibitor of phospholipase C (PLC). When expressed in CHO cells, a mutated α(2A)AR which has the amino and carboxyl termini of the third intracellular loop substituted with β2AR sequence poorly coupled to G(i) in adenylyl cyclase assays, and likewise displayed virtually no coupling to increased [Ca2+](i). These results all point toward a G(i)- versus a G(q)-mediated coupling pathway triggering release of intracellular calcium stores. The possibility that G(βγ) subunits released from α(2A)AR-G(i) coupling is the mechanism of PLC activation was explored in COS-7 cells by coexpressing α(2A)AR with the G(βγ) inhibitors transducin or a carboxy-terminal portion of the βAR kinase. Both βγ inhibitors markedly inhibited α(2A)AR modulation of [Ca2+](i) while not affecting thromboxane A2 receptor mediated stimulation of [Ca2+](i) via G(q) coupling. Thus, α(2A)AR couple to calcium release via G(i)-associated G(βγ) subunits. This coupling is present in multiple cell types and should be considered a major signal transduction pathway of this receptor.