We have developed a sensitive, specific solid-phase immunoradiometric assay (IRMA) of parathyroid hormone-related protein (PTH-RP) with use of affinity-purified polyclonal immunoglobulins. Antibodies recognizing PTH-RP(37-74) are immobilized to a polystyrene bead to "capture" analytes from the sample; antibodies to epitopes within the 1-36 amino acid region of PTH-RP are labeled with 125I. This IRMA recognizes PTH-RP(1-74) and PTH-RP(1-86) equivalently, but does not detect N-terminal or C-terminal fragments of PTH-RP, intact human parathyrin (PTH), or fragments of PTH. PTH-RP is not stable in plasma at 3-5 degrees C or room temperature, but a mixture of aprotinin (500 kallikrein units/L) and leupeptin (2.5 mg/L) improves PTH-RP stability in blood samples. In plasma collected in the presence of these protease inhibitors from normal volunteers and patients with various disorders of calcium metabolism, PTH-RP concentrations were above normal (greater than 1.5 pmol/L) in 91% (42 of 46) of patients with hypercalcemia associated with nonhematological malignancy. In plasma from patients with other hypercalcemic conditions (e.g., primary hyperparathyroidism, sarcoidosis, and vitamin D excess), PTH-RP was undetectable. Above-normal concentrations of PTH-RP and total calcium decreased to normal in a patient with an ovarian cyst adenocarcinoma after surgical removal of the tumor. We conclude that PTH-RP is related to and probably the causative agent of hypercalcemia in most patients with cancer, and that measurements of PTH-RP are useful in the diagnosis and management of patients with tumor-associated hypercalcemia.