The calcium-sensing receptor (CaR) controls parathyroid hormone (PTH) secretion and thus extracellular calcium (Ca2+o) homeostasis. CaR studies invariably employ simple buffers lacking most of the human plasma constituents capable of modulating CaR activity. These include aromatic amino acids and spermine (positive modulators) and albumin and phosphate that bind ionised Ca2+ in vivo thus inhibiting CaR. Since their serum concentrations change under various physiological and pathological conditions, here we aimed to model the likely CaR effects of such changes. CaR activity was determined as intracellular calcium (Ca2+i) mobilisation in Fura2-loaded CaR stably-transfected HEK-293 cells, by epifluorescence microscopy. Experimental Buffer contained (mM) 20 HEPES (pH 7.4), 125 NaCl, 4 KCl, 1 PO4, 0.5 CaCl2, 0.5 MgCl2 and 5.5 glucose. Chronic kidney disease (CKD) involves hyperphosphataemia and raised PTH secretion, but the causal mechanism of secondary hyperparathyroidism (SHPT) remains controversial. Here 3mM Ca2+o elicited robust, oscillatory Ca2+i mobilisation in the absence of phosphate, whereas co-treatment with 2mM phosphate abolished the response. Interestingly, in Ca2+o-free buffer where CaR was activated with neomycin (100μM), 2mM phosphate still inhibited CaR activity. Next, CaR calcium sensitivity was tested in the absence and presence of supplementation with 4% (w/v) bovine serum albumin, 1mM phosphate, 1mM MgCl2 and 100μM each of L-His, L-Phe, L-Trp and spermine (each equivalent to physiological concentrations). The EC50 for the Ca2+o-induced CaR response in experimental buffer (3.0 ± 0.3mM, S.E.M) was unchanged following supplementation (2.9 ± 0.1mM; N=4) suggesting that the endogenous supplements may have, in effect, cancelled each other out. However, further increasing the phosphate concentration to 2mM (as seen in CKD), caused a rightward shift in the concentration effect curve for Ca2+o (3.9 ± 0.3mM; P<0.05 by ANOVA, N=5). In contrast, doubling the spermine concentration to 200μM failed to alter CaR Ca2+o sensitivity (3.0 ± 0.2 mM; N=3) suggesting that physiological variations in blood spermine concentration may not affect CaR activity. In conclusion, while CaR experiments should take greater account of endogenous modulators, the current use of simple salt solutions produces appropriate EC50 values. In addition, while SHPT is often linked to low vitamin D levels or direct effects of phosphate on parathyroid cells, these data suggest that decreased CaR activation resulting from the hyperphosphataemia of CKD may play a much greater role than previously realised.