In order to investigate the possibility of the simultaneous expression of two different calcitonin genes during evolution, we studied the nature of calcitonin in: eel pericardium, salamander branchial region, rat thyroid, ox thyroid, medullary carcinoma of the human thyroid and human thyrotoxic goitre. Sensitive and specific RIAs for human calcitonin (hCT) and salmon calcitonin (sCT) were used in combination with high pressure liquid chromatography (HPLC). We found that two different immunoreactive calcitonins resembling hCT and sCT exist in eels, salamanders and rats. The major form is i-sCT-like in eels and salamanders, while it is i-hCT-like in rats. Extracts of ox thyroid contained, in addition to the major artiodactyl-CT, an i-hCT-like material. The various human thyroid extracts studied, contained i-hCT but no i-sCT-like material. This study demonstrates the simultaneous expression of two different calcitonin genes in several species. Taken with the presence of molecules resembling sCT and hCT in birds, this further evidence is consistent with duplication of the calcitonin gene early in vertebrate evolution.