Ligand binding by the calcium-sensing receptor (CaSR), which belongs to family C of the G-protein coupled receptor super-family, activates the phospholipase C-inositol triphosphate pathway and leads to an increase in intracellular calcium. CaSR inactivating mutations result in the hypercalcaemic disorders of familial benign hypocalciuric hypercalcaemia (FBHH) and neonatal severe primary hyperparathyroidism (NSHPT), whilst activating mutations result in the hypocalcaemic disorder of autosomal dominant hypocalcaemia with hypercalciuria (ADHH). We ascertained 51 probands (28 with FBHH, 6 with NSHPT, and 17 with ADHH) and investigated them for CaSR mutations. Venous blood was obtained with informed consent, using guidelines approved by the local ethical committee. Leucocyte DNA was extracted and used with CaSR-specific primers for PCR amplification. DNA sequence analysis of the PCR products revealed 25 mutations (8 in FBHH, 6 in NSHPT, and 11 in ADHH). The FBHH mutations consisted of R172G, R185Q, W208S, R220W, P221Q, S296N P748L and c-10 acg→atg; the NSHPT mutations consisted of R25X, C60F, Q164X, I212S, R392X and R680C; and the ADHH mutations consisted of A116P, E127G, C129Y, C131Y, P221L, E241K, N419S, E610G, T828N, S845N and 930delC. Functional characterisation of some CaSR mutations, by transient transfection into HEK293 cells, was performed to gain insights into the structure-function relationships of the CaSR. For example, the P221Q and P221L mutants, which are associated with FBHH and ADHH respectively, revealed that P221Q results in inactivation, with an EC50 value (mean±S.E.M.) of 4.43±0.18 mM, whilst P221L results in activation (EC50=0.87±0.15 mM), both of which significantly differed (P<0.01) from the wild-type CaSR (EC50=2.74±0.29 mM). Three-dimensional modelling based on the crystal structure of the rat metabotropic glutamate receptor and human CaSR amino-acid sequences, showed that P221 lies on the interface of the two monomers of the CaSR dimer in a cation-bound form and hence would play a pivotal role in CaSR ligand binding and activation.