Breast cancer cell metastases to bone result in osteolysis and release of large quantities of Ca2+ into the bone microenviroment. Extracellular Ca2+ (Ca(o)2+) acting through the Ca(2+)-sensing receptor (CaR), a member of G protein-coupled receptor superfamily, plays an important role in the regulation of multiple signaling pathways. Here, we find that expression of the CaR and Galpha(12) is significantly up-regulated in breast cancer cells (MDA-MB-231 and MCF-7) compared with nonmalignant breast cells (Hs 578Bst and MCF-10A). Ca(o)2+ induces a significant increase in extracellular [(3)H]phosphocholine (P-cho) production in breast cancer cells. Using an anti-CaR antibody to block Ca(o)2+ binding to the CaR and small interfering RNA (siRNA) to silence CaR gene expression, our data demonstrate that [(3)H]P-cho production in response to Ca(o)(2+)-stimulation is CaR-dependent. By analyzing cellular lipid profiles and using siRNA to silence choline kinase (ChoK) expression, we determine that the production of [3H]P-cho is primarily related to CaR-induced ChoK activation, and not degradation of choline phospholipids. Finally, by pretreatment of the cells with either pertussis toxin or C3 exoenzyme, co-immunoprecipiation of Galpha(i), Galpha(q) or Galpha12 with the CaR, and RhoA translocation, we found that the enhancement of ChoK activation and P-cho production in breast cancer cells occurs via a CaR-Galpha12-Rho signaling pathway.