The biosynthesis and release of calcitonin from chicken ultimobranchial glands were evaluated both in vivo and in vitro. Changes in the content of calcitonin in plasma and ultimobranchial glands of chicks during embryonic development were measured using a radioimmunoassay for salmon calcitonin that readily detects chicken calcitonin. Calcitonin content of the ultimobranchial glands underwent a striking rise during the late-embryonic and newborn periods, and a transient phase of brisk calcitonin secretion into blood was observed at the time of hatching. Ultimobranchial glands from late-embryo and newborn chicks, when incubated in vitro with radioactive leucine, incorporated labeled leucine into protein and released radioactive protein into the culture medium linearly over a period of 30 h. Analyses, by electrophoresis on polyacrylamide gels and by reactivity with anti-calcitonin sera, of radioactive protein released into the media revealed two radioimmunoassayable and immunoprecipitable peaks of chicken calcitonin. The two peaks could not be distinguished by their molecular weights upon electrophoresis on urea-SDS gels. These observations are most consistent with the existence of two isohormones of chicken calcitonin. No evidence for a biosynthetic precursor of calcitonin was obtained. Changes in calcium concentrations between 1.0 and 3.5 mM is incubation media in vitro did not affect release or storage of calcitonin, and changes in dietary calcium intake did not produce observable in vivo alterations in calcitonin content of ultimobranchial glands.