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 Ca2+. Recently, a calcium-sensing receptor (CaR), similar to the G protein-coupled receptor for external Ca2+ 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. 2. Whole-cell recordings in the permeabilized-patch configuration show that elevated extracellular Ca2+ concentration ([Ca2+]0) depolarizes these cells and induces oscillations in membrane potential. In voltage clamp, high [Ca2+]0 activates a cation conductance that underlies the depolarization. This conductance is cation selective, with a reversal potential near -25 mV indicating poor ion selectivity. 3. The CaR expressed in these cells is activated by other multivalent cations with a rank order potency of Gd3+ > Ba2+ > Ca2+ > > Mg2+. The insensitivity of these cells to high external Mg2+ contrasts with the reported sensitivity of the cloned CaR from parathyroid. 4. Elevation of [Ca2+]0 also stimulates increases in intracellular Ca2+ concentration ([Ca2+]i) and this effect is largely inhibited by the Ca2+ channel blocker nimodipine, indicating that L-type voltage-gated Ca2+ channels contribute to the response to elevated [Ca2+]0. 5. Elevated [Ca2+]0 induces an inward current under conditions where the only permeant external cation is Ca2+, indicating that influx via the cation conductance is another source of the increases in [Ca2+]i. 6. Extracellular Ca2+ stimulates 5-HT release with an EC50 of 1.5 mM. Nimodipine blocks 90% of the Ca2+0-induced 5-HT release, while other inhibitors of voltage-gated calcium channels had no effect. These data support an important role for L-type Ca2+ channels in CaR-induced hormone secretion. Although earlier studies indicate that high [Ca2+]0 induces release of Ca2+ from intracellular stores, thapsigargin-induced depletion of these stores did not affect secretion from these cells, indicating that Ca2+ influx is necessary and sufficient for the Ca2+0-induced 5-HT secretion. 7. Inhibition of protein kinase C (PKC) using chelerythrine, staurosporine, or calphostin C inhibited Ca2+0-induced 5-HT release by 50% while phorobol 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 [Ca2+]0. However, pretreatment with D609, a selective inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), inhibited Ca(2+)-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 [Ca2+]0.