Vascular calcification (VC) is highly correlated with increased morbidity and mortality in chronic kidney disease (CKD) patients. Allosteric modulation of the calcium sensing receptor (CaSR) by the calcimimetics decreases serum parathyroid hormone, calcium and phosphorus (1), and can inhibit VC in animal CKD models (2). However, it is unknown whether the anti-VC effect is mediated by direct action on vascular smooth muscle cells (VSMC). Here we have examined VSMC CaSR expression, CaSR function in regulating mineralized matrix deposition, and the effects of R-568 on VSMC mineral deposition. Human and bovine VSMC were isolated and cultured in 10% FCS-DMEM containing 1.2mM free ionized extracellular calcium concentration ([Ca²⁺]). CaSR expression was determined by Western blotting and immunofluorescence. Mineral deposition was assessed by alizarin red staining and ⁴⁵Ca incorporation. VSMC CaSR expression was demonstrated by Western blotting of cell lysates and immunfluorescence staining of cultured cells. Incubation of confluent VSMC with increasing extracellular [Ca²⁺] for 24 hours decreased CaSR expression. Western blotting during osteogenic differentiation demonstrated marked CaSR downregulation when mineralized nodules were formed. Acute (5 min) stimulation of VSMC with 5mM Ca²⁺ or 50μM Gd³⁺, a non-permeable agonist, significantly stimulated ERK phosphorylation, suggesting a functional CaSR. Culturing cells with 1.8 or 2.5mM Ca²⁺ or 10 or 50μM Gd³⁺ enhanced (P<0.05) mineral deposition compared with VSMC cultured with 1.2mM Ca²⁺. R-568 (1nM) reduced nodule formation and significantly inhibited mineralisation by cells cultured with 1.8mM Ca²⁺ (P<0.001). These results demonstrate that VSMC express a functional CaSR that is down-regulated with mineralized matrix deposition. In line with previously reported data (2), there is an association between calcimimetic use and reductions in VC. These data suggest that the potential inhibitory effect of calcimimetics on VC is mediated by the CaSR. The nature of this mechanism is currently under investigation.