1. Expression of receptors to extracellular calcium enables parafollicular cells of the thyroid gland (PF cells) to release calcitonin (CT) and serotonin (5_HT) in response to increased external Ca¥. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled receptor for external Ca¥ cloned from parathyroid gland, was shown to be expressed in PF cells. Using a highly purified preparation of sheep PF cells, we have examined the electrical and biochemical processes coupling CaR activation to hormone release. cation is Ca¥, indicating that influx via the cation conductance is another source of the increases in [Ca¥]é. 6. Extracellular Ca¥ stimulates 5_HT release with an ECÛÑ of 1·5 mÒ. Nimodipine blocks 90% of the Cafi-induced 5_HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca¥ channels in CaR-induced hormone secretion. Although earlier studies indicate that high [Ca¥]ï induces release of Ca¥ from intracellular stores, thapsigargin-induced depletion of these stores did not affect secretion from these cells, indicating that Ca¥ influx is necessary and sufficient for the Cafi-induced 5_HT secretion. 7. Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Cafi-induced 5_HT release by 50% while phorbol ester-induced 5_HT secretion was completely inhibited. Thus, PKC is an important component of the pathway linking CaR activation to hormone release. However, another as yet unknown second messenger also contributes to this pathway. 8. We tested the contribution of two different phospholipases to the CaR responses to determine the source of the PKC activator diacylglycerol (DAG). Selective inhibition of phosphatidylinositol-specific phospholipase C (PI-PLC) with U73122 had no effect on the response to elevated [Ca¥]ï. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca¥-induced 5_HT release to 50% of control indicating that phosphatidylcholine is a likely source of DAG in the response of PF cells to elevated [Ca¥]ï.