The extracellular calcium-sensing receptor (CaR) inhibits parathyroid hormone secretion and renal calcium reabsorption to prevent hypercalcaemia however, the precise nature of the intracellular signals controlling this remain to be elucidated (Ward, 2004). Previous studies report an association of the CaR with the cytoskeletal protein filamin (Hjalm et al. 2001) and therefore here we examined the consequence of CaR activation on the cytoskeletal structure and cell morphology of human embryonic kidney-293 cells stably transfected with CaR (CaR-HEK; NPS Pharmaceuticals, UT, USA). For each treatment, 4 dishes of cells were incubated in serum-free DMEM medium for up to 3 h and then for each dish, 3 regions of cells were imaged digitally for morphological assessment. Alternatively cells were exposed to various CaR agonists at 37^oC for 5 mins, in HEPES buffer ((mM): 20 HEPES (pH 7.4), 125 NaCl, 4 KCl, 0.5 CaCl₂, 0.5 MgCl₂ and 5.5 glucose), lysed in RIPA buffer and then phosho-ERK content was determined by semi-quantitative immunoblotting and densitometry (Ward et al. 2002). Incubation of the cells in serum-free medium induced cell stellation, whereas cotreatment with the calcimimetic (CaR positive allosteric modulator) NPS-467R (0.1-1μM) or exposure to elevated extracellular Mg²⁺ levels (1-10mM; Mg²⁺_o) elicited dose-dependent cell rounding with process retraction (>90% reduction in process number). This effect was detectable within 1 h and was sustained for at least 3 h following the removal of agonist. These treatments were without effect in vector-transfected HEK cells, and, in CaR-HEK cells, the calcimimetic effect was stereoselective since NPS-467S (1μM) failed to alter CaR-HEK cell morphology. To confirm that the responses to NPS-467R and high [Mg²⁺]_o are mediated by the CaR, we cotreated the cells with the novel CaR “antagonist” (calcilytic; negative allosteric modulator) NPS-89636 (1μM) and found that the cotreatment abolished the responses. This drug also blocked ERK activation in response to increased extracellular Ca²⁺ concentration (4mM; control, 1.0 ± 0.5 arbitrary densitometry units ± SEM; 4mM Ca²⁺, 34.5 ± 17.2, P<0.001 by ANOVA; 4mM Ca²⁺ plus calcilytic 2.5 ± 1.3, P<0.001 vs 4mM Ca²⁺ only; n=4), or to the CaR agonists Gd³⁺ (60μM) and neomycin (100μM) confirming its inhibitory action on the CaR. Cotreatment with the rho-kinase inhibitor Y27632 (10μM) also attenuated CaR-induced cell rounding indicating that the response is most likely mediated via the small G protein rho. Together, these data demonstrate that the filamin-coupled CaR can elicit rho kinase-mediated morphological changes, raising the possibility that cytoskeletal changes may contribute, at least in part, to CaR function.