A cytochemical bioassay for parathyroid hormone (PTH) was used for the characterization of the biological activity of circulating forms of the hormone. PTH-stimulated glucose-6-phosphate dehydrogenase activity in distal convoluted tubule cells was quantitated by integrating microdensitometry and the response to native bovine (b)PTH(1-84) was found to be linear between graded doses of hormone from 5 fg/ml to 5 pg/ml. Synthetic bPTH(1-34) and human (h)PTH(1-34) elicited a parallel and equimolar response; however calcitonin, ACTH, glucagon, epinephrine, vasopressin, and insulin failed to significantly stimulate the enzyme in doses up to 100,000 times greater than the lowest concentration of bPTH used. The assay was capable of distinguishing hormonal activity in normal, hypoparathyroid, and hyperparathyroid human plasma. After gel chromatography, bioactivity in plasma of hyperparathyroid patients with skeletal disease but normal kidney function coeluted mainly with bPTH(1-84), whereas bioactivity in plasma of hyperparathyroid patients with skeletal disease but severe uremia coeluted in approximately equivalent amounts with bPTH(1-84) and hPTH(1-34). Despite the abundance of small molecular-weight bioactivity in the peripheral circulation in uremia, approximately 85% of the bioactivity in the parathyroid venous effluent coeluted with bPTH(1-84). The results therefore demonstrate the sensitivity and specificity of the assay for PTH and its utility in measuring the hormone in human parathyroid disorders. The results furthermore demonstrate the importance of entities cochromatographing with bPTH(1-84) in comprising the circulating bioactive hormone in hyperparathyroidism, and support the concept of a biological role for smaller forms of PTH, at least in chronic renal failure.