Calcitonin (CT) is secreted mainly by parafollicular C cells. In normal subjects, serum CT (sCT) levels are low and barely detectable by commonly used assays. Increased sCT levels are highly suggestive of medullary thyroid carcinoma (MTC). Since the clinical manifestation of MTC is a thyroid nodule, either single or in the context of a multinodular goitre, the routine measurement of sCT in the evaluation of thyroid nodule(s) facilitates the diagnosis of MTC. sCT measurement is indeed more sensitive than cytology in finding MTC. There are two major benefits from this clinical practice: (a) the surgeon is alerted to the need to perform total thyroidectomy and central compartment lymphadenectomy, which is the minimal surgical treatment for MTC; and (b) the outcome of MTC is favourably affected because it is usually identified at a less advanced stage. However, other non-MTC causes of hypercalcitoninaemia and false sCT positivity do exist and must be recognized. The differential diagnosis is possible using previously validated assays against possible sources of interference, and performing stimulation tests with pentagastrin or, eventually, calcium infusion.