Small elevations of extracellular calcium cause vasodilation in blood vessels via an endothelium-dependent mechanism. Recently, functional expression of a receptor that senses changes in extracellular calcium, the calcium-sensing receptor (CaSR), has been demonstrated in vascular tissue (Weston et al., 2005). Particularly, functional evidence was presented for the existence of an endothelial CaSR, stimulation of which caused smooth muscle hyperpolarisation. Thus, we investigated the molecular and functional expression of the CaSR in primary isolated vascular endothelial cells. Rat aortic endothelial cells (RAEC) were obtained by an explant technique as previously described (Suh et al., 1999). Cells obtained using this technique displayed characteristic cobblestone morphology when confluent, were positively immunostained for the von Willebrand factor and fluoresced following incubation in DiL-Ac-LDL. Immunocytochemical studies using a monoclonal antibody against the CaSR confirmed the presence of the receptor protein. In functional studies, RAEC were loaded with the calcium indicator dyes FURA-2 or INDO-1 and changes in intracellular calcium concentration examined during application of known CaSR agonists. Raising extracellular calcium (from 0.5 mM to 1.5 and 3 mM) produced concentration-dependent elevations in intracellular calcium. These responses were mimicked by the CaSR activators 0.8 mM Mg²⁺, 200 μM neomycin, and 500 μM gadolinium. Responses to agonists varied between cells, and included large and sustained increases and high-frequency oscillations, suggesting a heterogeneity of cellular expression and/or function of the receptor. We conclude that the vascular endothelium expresses a functionally active CaSR, activation of which elevates intracellular calcium. Since this further modulates smooth muscle membrane potential to bring about hyperpolarisation (Weston et al 2005) and thus vasodilation, the CaSR may present a useful therapeutic target for the treatment of hypertension.