Parathyroid specimens removed from patients with clinical hyperparathyroidism were cultured in a 2-layer soft-agar system. Four patients had parathyroid hyperplasia and 1 had a parathyroid adenoma. Colonies grew from single-cell suspensions of each specimen. Plating efficiency ranged from 0.001-0.05%. No colonies grew from 2 normal bovine parathyroid specimens. Parathormone was detected in 0.9% NaCl solution incubated with the culture plates of 3 of the 4 human specimens tested. Parathormone levels determined by radioimmunoassay ranged from 10.4 > 100 ng/ml. Plates tested serially showed a progressive rise in parathormone levels with time and an increase in colony size and number. Microscopic evaluation of the cellular layer showed clusters of cells morphologically consistent with parathyroid origin. Colonies remained viable for .apprx. 3 wk. Malignancy of tissue in vivo is not necessary for colony formation in agar and human parathyroid hyperplasia or adenoma cells produce and secrete parathormone in this system.