Calcitonin (CT) secretion by parafollicular cells of the thyroid (C cells) is regulated by small changes in the concentration of extracellular calcium ([Ca2+]e). Elevation of [Ca2+]e elicits a rise in the C cell cytoplasmic calcium concentration and stimulates CT release. The molecular entity through which C cells detect changes in [Ca2+]e and modulate hormone secretion is unknown. Recently, an extracellular calcium-sensing receptor (CaR) complementary DNA was isolated from bovine parathyroid gland. To assess whether parathyroid cells and C cells use similar mechanisms to detect changes in ambient Ca2+, rat, human, and sheep C cells were examined for expression of the parathyroid CaR or a related receptor isoform. Reverse transcription-polymerase chain reaction analysis identified CaR transcripts in rat and human thyroid gland. Northern blot analysis demonstrated CaR messenger RNA (mRNA) in rat thyroid gland, a human medullary thyroid carcinoma (MTC) isolate, and a highly enriched preparation of sheep C cells. Rat MTC 44-2 cells, a cell line responsive to changes in [Ca2+]e, express abundant levels of CaR mRNA. Human TT cells, a C cell line lacking the extracellular calcium-sensing function, have undetectable levels of CaR mRNA by Northern blot analysis. Western blot analysis, using antiserum specific to the parathyroid CaR, detected CaR protein in rMTC 44-2, but not TT cells. Immunostaining of both dispersed sheep C cells and rat thyroid gland sections identified C cell-specific expression of the CaR protein, and in situ hybridization analysis confirmed the C cell-specific expression of CaR mRNA in the intact rat thyroid. The nucleotide sequence of the coding region of the rMTC 44-2 CaR transcripts was found to encode the same CaR protein as that expressed in the parathyroid and kidney. The results demonstrate that C cells express the same extracellular calcium-sensing receptor that is found in parathyroid and kidney, and the presence of this receptor protein in C cell lines correlates with the extracellular calcium-sensing function. This CaR is likely to represent the primary molecular entity through which C cells detect changes in [Ca2+]e and control CT release, suggesting that activation of the same receptor can either stimulate or inhibit hormone secretion in different cell types.