Background: There is increasing evidence that the calcium-sensing receptor (CaR) is expressed in the vasculature; in endothelial cells (1), vascular smooth muscle cells (VSMC’s) (2) and perivascular neurones (3). However the function and underlying mechanisms of CaR-mediated processes on vascular tone remain unclear. The present work investigates CaR-mediated mechanisms that influence vascular toneMethods: New Zealand White rabbits (2-3kg) were killed by iv. injection of sodium pentobarbitone (120mg kg-1) in accordance with Schedule I, UK Animals Scientific Procedures Act, 1986. Segments of 2nd-order branches of the superior mesenteric artery were mounted in a wire myograph and maintained at 37°C in gassed (95% O2/5%CO2) Krebs-Henseleit solution (1mM [Ca2+]o). Arteries were precontracted with 10µM methoxamine and cumulative additions of CaCl2 (1.5-6mM) were added in the absence and presence of the inhibitors tested. Functional endothelium was tested using 10µM carbachol and was removed mechanically using a human hair. Results are given as means ±S.E. and were analysed by 2-way ANOVA and Bonferroni post-hoc tests; n=5-7 animals.Results: Increasing [Ca2+]o from 1.5-6mM induced concentration-dependent vasorelaxations that were abolished with the removal of the endothelium (38.2±12.8% at 2mM and 93.1±3.2% at 6mM). The calcilytic Calhex-231 (3µM) attenuated the Cao2+ -induced vasorelaxations, confirming the involvement of CaR. 300μM L-NAME (nitric oxide synthase inhibitor), 10µM ODQ (soluble guanylate cyclase (GC) inhibitor) and 1µM KT5823 (PKG inhibitor) all effectively abolished Cao2+-induced vasorelaxations. 10mM TEA (non-specific K+ channel blocker) and 100nM iberiotoxin (BKCa blocker) attenuated the relaxation (52.3±6% and 52.9±7% at 6mM respectively) and 10µM linopirdine (Kv7 blocker) reduced maximum relaxation by ~20%. When 100nM charybdotoxin (IKCa/BKCa blocker) was added together with 100nM iberiotoxin, attenuation of the relaxation was augmented. 100nM Apamin (SKCa blocker), 10µM glibenclamide (KATP blocker) and 10µM indomethacin (COX inhibitor) had no effect.Conclusion: These results suggest that CaR mediates endothelium-dependent vasorelaxations via the Nitric oxide-GC-PKG pathway which activates BKCa and Kv7 channels on the underlying VSMC’s. Concurrently, CaR stimulation leads to endothelial IKCa activation, augmenting the vasorelaxations. A significant Cao2+-induced relaxation was observed at 2mM [Ca2+]o, indicating that physiologically, serum Ca2+ activates luminal endothelial CaRs, leading to a basal nitric oxide release/IKCa activation that helps regulate vascular tone. CaR may also represent a novel drug target in the treatment of hypertension