Calcitonin and its carboxyl-terminal flanking peptide (PDN-21), also encoded by the calcitonin gene, were measured by RIA in unextracted serum of normal subjects and patients with primary hyperparathyroidism and surgically verified and suspected medullary thyroid carcinoma. Serum PDN-21 was detectable (greater than 0.005 ngeq/ml) in the large majority of normal subjects (92%), and the values increased significantly more in men than women (4.8- and 2.0-fold, respectively; P less than 0.01) in response to 1-min iv calcium injections. Calcitonin was detectable (greater than 0.025 ngeq/ml) in only 25% of normal subjects before iv calcium and became measurable after iv calcium in 88% of men and 41% of women. In patients with chronic hypercalcemia due to primary hyperparathyroidism, PDN-21 and calcitonin were within normal limits. In normal subjects, iv pentagastrin (0.5 microgram/kg BW) did not increase PDN-21, and calcitonin remained undetectable. In 41 medullary thyroid carcinoma patients, basal PDN-21 and calcitonin levels were increased similarly, and they were stimulated in response to iv calcium or iv pentagastrin. In 5 siblings of medullary thyroid carcinoma patients, PDN-21 and calcitonin were increased in response to iv pentagastrin, and we suspect C-cell hyperplasia or medullary thyroid carcinoma. In conclusion, a diagnostically useful RIA for the measurement of PDN-21 in unextracted serum which complements calcitonin measurements has been developed.