A new peptide spanning residues 28-54 of human parathyroid hormone (PTH) was synthesized and used to develop a homologous immunoradiometric assay specific for the mid-region of human PTH. The peptide was coupled to cellulose and used to absorb mid-region antibodies from a goat antiserum against intact human PTH. This assay has been applied to the measurement of circulating PTH in man: in normal subjects the concentration in serum ranged from undetectable (less than 40 pg/ml) to 70 pg/ml, the reference standard being the human PTH 28-54 peptide. In patients with primary hyperparathyroidism concentrations ranged from 120 to 1800 pg/ml. Hormone was not detected in patients with hypoparathyroidism. In normal subjects and in patients with primary hyperparathyroidism the mid-region PTH concentrations were similar to those obtained in an amino-terminal specific assay. By contrast, carboxy-terminal PTH concentrations were markedly higher being 10-fold greater in both groups studied. In patients with primary hyperparathyroidism undergoing parathyroidectomy and in chronic renal failure patients who were infused with calcium, mid-region and amino-terminal PTH disappeared much more rapidly than carboxy-terminal PTH. However, although mid-region PTH was initially cleared as quickly as amino-terminal PTH, it then reached a plateau and remained at a higher level. Thus the mid-region specific assay described here is proving to be of value in the study of the secretion and metabolism of PTH.