The extracellular calcium-sensing receptor (CaR) senses small fluctuations of the extracellular calcium (Ca2+e) concentration and translates them into potent changes in parathyroid hormone secretion. Dissecting the regulatory mechanisms of CaR-mediated signal transduction may provide insights into the physiology of the receptor and identify new molecules as potential drug targets for the treatment of osteoporosis and/or hyperparathyroidism. CaR can be phosphorylated by protein kinase C (PKC) and G protein-coupled receptor kinases (GRKs), and has been shown to bind to β-arrestins, potentially contributing to desensitization of CaR, although the mechanisms by which CaR-mediated signal transduction is terminated are not known. We used a PKC phosphorylation site-deficient CaR, GRK and β-arrestin overexpression or down-regulation to delineate CaR-mediated desensitization. Fluorescence-activated cell sorting was used to determine whether receptor internalization contributed to desensitization. Overexpression of GRK 2 or 3 reduced Ca2+e-dependent inositol phosphate accumulation by more than 70%, whereas a GRK 2 mutant deficient in Gαq binding (D110A) was without major effect. Overexpression of GRK 4–6 did not reduce Ca2+e-dependent inositol phosphate accumulation. Overexpression of β-arrestin 1 or 2 revealed a modest inhibitory effect on Ca2+e-dependent inositol phosphate production (20–30%), which was not observed for the PKC phosphorylation site-deficient CaR. Agonist-dependent receptor internalization (10–15%) did not account for the described effects. Thus, we conclude that PKC phosphorylation of CaR contributes to β-arrestin-dependent desensitization of CaR coupling to G proteins. In contrast, GRK 2 predominantly interferes with G protein-mediated inositol-1,4,5-trisphosphate formation by binding to Gαq.