The C family G-protein-coupled receptors contain members that sense amino acid and extracellular cations, of which calcium-sensing receptor (CASR) is the prototypic extracellular calcium-sensing receptor. Some cells, such as osteoblasts in bone, retain responsiveness to extracellular calcium in CASR-deficient mice, consistent with the existence of another calcium-sensing receptor. We examined the calcium-sensing properties of GPRC6A, a newly identified member of this family. Alignment of GPRC6A with CASR revealed conservation of both calcium and calcimimetic binding sites. In addition, calcium, magnesium, strontium, aluminum, gadolinium, and the calcimimetic NPS 568 resulted in a dose-dependent stimulation of GPRC6A overexpressed in human embryonic kidney cells 293 cells. Also, osteocalcin, a calcium-binding protein highly expressed in bone, dose-dependently stimulated GPRC6A activity in the presence of calcium but inhibited the calcium-dependent activation of CASR. Coexpression of beta-arrestins 1 and 2, regulators of G-protein signaling RGS2 or RGS4, the RhoA inhibitor C3 toxin, the dominant negative Galpha(q)-(305-359) minigene, and pretreatment with pertussis toxin inhibited activation of GPRC6A by extracellular cations. Reverse transcription-PCR analyses showed that mouse GPRC6A is widely expressed in mouse tissues, including bone, calvaria, and the osteoblastic cell line MC3T3-E1. These data suggest that in addition to sensing amino acids, GPRC6A is a cation-, calcimimetic-, and osteocalcin-sensing receptor and a candidate for mediating extracellular calcium-sensing responses in osteoblasts and possibly other tissues.