The mechanism by which the calcium-sensing receptor (CaR) regulates parathyroid hormone (PTH) secretion to control calcium homeostasis remains unclear. Orthosteric CaR activation with high extracellular Ca²⁺ (Ca²⁺_o) concentration, or allosteric CaR activation with the calcimimetic NPS R-467, elicits rho/rho kinase-mediated cell process retraction (1). However, the aromatic amino acids (allosteric CaR activators) are without effect despite eliciting intracellular calcium (Ca²⁺_i) mobilisation. To examine the CaR agonist concentration-dependency of actin polymerisation (after 3 hours) relative to acute changes in [Ca²⁺]_i and ERK activation, we examined HEK-293 cells stably transfected with CaR (CaR-HEK) using phalloidin-TRITC, fura-2 and phospho-ERK antibody respectively. Ca²⁺_o caused a concentration-dependent increase in actin polymerisation with an EC₅₀ of 2.1 mM (peaking at 3mM), but stimulated ERK activation and Ca²⁺_i less potently (EC₅₀, 3.7 and 3.4 mM respectively). Extracellular magnesium also elicited greater potency for actin polymerisation (EC₅₀, 4.1 mM; peaking at 6mM) than for ERK activation (EC₅₀, 8.2 mM). Also, NPS R-467 elicited actin polymerisation, in the presence of 1.2mM Ca²⁺_o (i.e. the plasma free ionised Ca²⁺_i concentration) with an EC₅₀ of 73nM, whereas even 1μM NPS R-467 failed to elicit ERK activation without a further increase in the baseline Ca²⁺_o concentration. In contrast, L-Phe, L-Trp and L-His (10mM) each failed to alter actin polymerisation in the presence of 1.2 mM Ca²⁺_o. The aromatic amino acids have been reported to elicit CaR-induced Ca²⁺_i mobilisation via rho with Ca²⁺_o responses mediated via phospholipase C (2). However, we found that the PLCβ inhibitor U73122 (1μM) substantially inhibited both L-Phe (10mM) and Ca²⁺_o (3mM)-induced Ca²⁺_i responses. Furthermore, C3 exoenzyme pre-treatment had no effect on L-Phe-induced Ca²⁺_i mobilisation despite abolishing high Ca²⁺_o-induced process retraction. We have proposed a mechanism of CaR-regulated PTH secretion by which Ca²⁺_o concentration may alter the cell cytoskeleton to modulate the trafficking of PTH-bearing secretory vesicles (1). Thus, here we show that the CaR stimulates actin polymerisation more potently than for PLC-mediated Ca²⁺_i mobilisation or ERK activation and over a range of Ca²⁺_o concentrations much closer to those seen physiologically. However, under the conditions tested, L-Phe failed to elicit CaR-induced actin polymerisation. Furthermore, contrary to previous reports (2) L-Phe-induced Ca²⁺_i mobilisation could be attenuated by PLCβ inhibition but not by rho inactivation.