Extracellular Ca²⁺ homeostasis is maintained by the suppressive action of the calcium-sensing receptor (CaR) on parathyroid hormone secretion and renal Ca²⁺ reabsorption. Substitution of the CaR putative protein kinase C (PKC) consensus sequence, Thr-888, alters significantly the function of the receptor expressed in HEK-293 cells, whilst the CaR-induced suppression of PTH secretion is modulated by treatment with phorbol esters and PKC inhibitors. Therefore, to investigate CaR phosphorylation directly we developed an affinity-purified, phospho-specific polyclonal antibody that recognises the phosphorylated form of CaR_T888. Using this antibody, we examined PKC-mediated CaR_T888 phosphorylation in human CaR-transfected HEK cells by immunoblotting and immunofluorescence. Acute treatment of the cells with the phorbol ester PMA increased the immunoreactivity of two bands corresponding to the immature (140kDa) and mature (160kDa) glycosylated CaR and increased total cellular immunoreactivity by immunofluorescence. These signals could be blocked selectively using the phosphorylated but not the non-phosphorylated immunising peptide and were absent in cells expressing a mutant CaR lacking Thr-888 (CaR_T888A). Increasing the concentration of extracellular Ca²⁺ (Ca²⁺_o) from 0.5 to 2.5mM induced a small rise (+26±18%, ±sem) in CaR_T888 phosphorylation. Increasing it to 5mM, or, addition of the calcimimetic (i.e. CaR positive allosteric modulator) NPS-467R (1μM; +42±10%, P<0.05 ANOVA, N=4) further elevated the response, the effect of NPS-467R being stereoselective. The CaR agonist-induced increase in CaR_T888 phosphorylation was localised to the cell membrane as demonstrated by confocal microscopy and was sensitive to PKC inhibition using GF109203X (-60±3%, P<0.001). No responses were seen in empty vector-transfected HEK cells. Furthermore, in Fura2-loaded CaR-HEK cells, phorbol ester treatment abolished 2.5mM Ca²⁺_o-induced Ca²⁺_i oscillations while GF109203X, or chronic PMA pre-treatment, converted the oscillatory response to a sustained plateau response. In addition, the agonist-sensitivity of CaR_T888A was enhanced relative to wild-type CaR. Finally, following the removal of an acute PMA pretreatment, the CaR_T888 residue was dephosphorylated 61% (±13%, P<0.05) within 5 min, a process that was completely blocked by the protein phosphatase 1/2A inhibitor calyculin (100nM). Together these data prove that the CaR can be phosphorylated by PKC on residue Thr-888 and that agonist stimulation of the receptor elicits feedback phosphorylation of this residue. By this mechanism it may be possible to alter the physiological sensitivity of the CaR and thus affect calcium homeostasis.