Extracellular Ca2+ homeostasis is maintained by the action of the Ca2+-sensing receptor (CaR) on parathyroid hormone (PTH) secretion and renal Ca2+ reabsorption. CaR contains several protein kinase C (PKC) consensus sequences of which one, CaRT888, is the key site responsible for PKC-mediated inhibition of CaR-elicited Ca2+i mobilisation in vitro and PTH secretion in vivo. However, we have not yet identified the PKC isotype responsible for CaRT888 phosphorylation nor determined whether CaR signalling can be modulated by cAMP given the fundamental regulatory roles of these second messengers in PTH secretion. Therefore, siRNAs were used to knockdown specific PKC isoforms in turn, in CaR-transfected HEK-293 cells (CaR-HEK) with the effect on Ca2+i mobilisation then determined in Fura2-loaded cells by epifluorescence microscopy. Experimental buffer contained (mM) 20 HEPES (pH 7.4), 125 NaCl, 4 KCl, 0.5 CaCl2, 0.5 MgCl2 and 5.5 glucose. Following the siRNA-induced knockdown of PKCα, the EC50 of the CaR for Ca2+o was significantly lowered (3.2 ± 0.1 mM PKCα siRNA vs. 3.9 ± 0.2 mM siRNA control transfection; P<0.001, n=9) indicating that PKCα contributes to the inhibition of CaR-induced Ca2+i mobilisation. In contrast, no significant effect on CaR responsiveness to Ca2+o was observed following transfection with siRNAs against PKCs β, δ, ε or θ. In addition, PKCα knockdown lowered the threshold for Ca2+o-induced CaR activation whereby 1.8 mM Ca2+o elicited Ca2+i mobilisation in CaR-HEKs transfected with siRNA for PKCα but not with control siRNA (P<0.01; n=6). Intriguingly, this suggests that PKCα may even be active before the CaR elicits detectable Ca2+i mobilisation. Indeed, a similar effect was seen using the PKC inhibitor GF109203X (1μM) which permitted significant responses to 2mM Ca2+o that were not observed in the inhibitor’s absence (P<0.05, n=7). Next, following 2.5mM Ca2+o-induced CaR stimulation, cotreatment with either forskolin (10μM; adenylate cyclase activator), IBMX (75μM; 3-isobutyl-1-methylxanthine, phosphodiesterase inhibitor) or isoprenaline (100nM; β-adrenoceptor agonist) significantly enhanced the resulting Ca2+i mobilisation. In contrast, the inactive analogue 1,9 dideoxyforskolin (10μM) was without effect. Indeed forskolin (10μM) also reduced the agonist concentration threshold for CaR activation whereby 1.8mM Ca2+o elicited Ca2+i mobilisation in the presence of forskolin but not in its absence (P<0.001, n=9). Together these data demonstrate that in CaR-HEK cells, PKCα inhibits CaR-induced Ca2+i mobilisation including at subthreshold agonist concentrations. In contrast, cAMP lowers the threshold for CaR-induced Ca2+i mobilisation demonstrating the potential of the CaR to provide integration of multiple intracellular signals in Ca2+o homeostasis.