Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca(2+)) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca(2+) concentrations increased from 6.2+/-0.3mg/dL to 9.9+/-0.5mg/dL (4 min; P<0.01). Serum CT increased from 16.7+/-8.0 pg/mL to 87.1+/-55.8 pg/mL at 2 min, and 102.5+/-51.1 pg/mL at 4 min (P<0.01). Serum CT returned to baseline by 20 min, whereas serum Ca(2+) returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca(2+) or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca(2+) concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.