GPRC6A displays high sequence homology to the Ca2+-sensing receptor (CaSR). Here we report that the calcimimetic Calindol and the calcilytic NPS2143 antagonize increases in inositol phosphate elicited by L-ornithine-induced activation of mouse GPRC6A after transient coexpression with Galpha(qG66D) in HEK293 cells. The calcilytic Calhex 231 did not modulate this response. A three-dimensional model of the GPRC6A seven transmembrane domains (TMs) was constructed. It was used to identify seven residues strictly conserved within the CaSR and GPRC6A allosteric binding pockets, and previously demonstrated to interact with calcilytics or calcimimetics. The mutations F666A(3.32), F670A(3.36), W797A(6.48) caused a loss of L-ornithine ability to activate GPRC6A mutants. The F800A(6.51) mutant was not implicated in either Calindol or NPS 2143 recognition. The E816Q(7.39) mutation led to a loss of Calindol antagonist activity but was without effect on NPS2143 inhibitory response. In summary, these data suggest that Calindol is primarily anchored through an H-bond to E816(7.39) in TM7 and highlight important local differences at the level of the CaSR and GPRC6A allosteric binding pockets. We have identified the first antagonists of GPRC6A that could represent new tools to analyze GPRC6A functions and serve as chemical leads for the development of more specific modulators.