The calcium-sensing receptor (CaR) regulates parathyroid hormone secretion and renal Ca²⁺ reabsorption and thus controls Ca²⁺_o homeostasis. CaR_T888 is a key intracellular signalling determinant whose phosphorylation by protein kinase C inhibits CaR-elicited Ca²⁺_i mobilisation [1,2]. Here, we examined the concentration-dependence of Ca²⁺_o on CaR_T888 phosphorylation by semi-quantitative immunoblotting [2]. HEK-293 cells stably expressing human CaR (CaR-HEK) were incubated for 10mins at 37^oC in Hepes-containing buffer. Raising [Ca²⁺]_o from 0.5 to 2.5mM caused a 5.5-fold increase in CaR_T888 phosphorylation (160kDa mature, membrane-localised receptor; P<0.001 by ANOVA, N=6). However, following 10min treatment with higher Ca²⁺_o concentrations (3-5mM), CaR_T888 phosphorylation was not elevated. The effect of 2mM Ca²⁺_o treatment (which also elicited oscillatory Ca²⁺_i mobilisation) on CaR_T888 phosphorylation was sustained for at least 20mins. However, 5mM Ca²⁺_o (associated with sustained Ca²⁺_i mobilisation) did increase CaR_T888 phosphorylation briefly, but with levels returning to baseline within 2mins suggesting that increased CaR activation might induce subsequent CaR_T888 dephosphorylation. To test this, CaR_T888 phosphorylation was first induced by phorbol ester treatment (1μM PMA, 10 mins) with the cells then incubated for 30secs in buffer containing either 1.2, 2.2 or 5mM Ca²⁺_o or 1.2mM Ca²⁺_o plus the CaR positive allosteric modulator L-Phe (10mM) [3]. Increasing [Ca²⁺]_o stimulated subsequent CaR_T888 dephosphorylation (1.2mM,33±14%; 2.2mM, 52±9%; 5mM, 78±5%, P<0.05 vs 1.2 by ANOVA; N=8) whereas L-Phe was without additional effect (31±9%). Therefore, orthosteric CaR activation elicits CaR_T888 dephosphorylation perhaps contributing to the high frequency oscillatory and sustained Ca²⁺_i mobilisation responses to Ca²⁺_o. In contrast, the failure of L-Phe to stimulate CaR_T888 dephosphorylation may explain why this CaR modulator elicits low frequency Ca²⁺_i oscillations. Therefore, CaR_T888 is subject to agonist-specific phosphorylation and dephosphorylation in CaR-HEK cells. Such receptor-elicited dephosphorylation of this protein kinase C consensus site could help explain the control of signal oscillation frequency observed in this and other type III G protein-coupled receptors.