Medullary thyroid carcinoma (MTC) is associated with amyloid deposition in the surrounding tissues. MTC-positive tumor thyroid tissues surgically removed from patients were used in our study to extract amyloid. We tested the MTC extracts for the presence of amyloid by measuring fold enhancement of thioflavin T fluorescence. Transmission electron microscopic study and atomic force microscopy of MTC patient extracts revealed typical amyloid fibrils. Matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis demonstrated full-length calcitonin as the constituent of the MTC amyloid from seven patients. Our results unequivocally demonstrated that full-length calcitonin is the sole constituent of amyloid in MTC.