The question of parathyroid autonomy in primary hyperparathyroidism has been the subject of conflicting immunoassay data. We studied the effects of calcium infusion (12 mg/kg/3h) and calcium injection (3 mg/kg/10 min) on peripheral plasma parathyroid hormone (iPTH) determined with a multivalent antiserum and on the excretion of nephrogenous cyclic AMP in normal subjects and in 7 patients with primary hyperparathyroidism who displayed only mild, intermittent hypercalcemia. In control subjects, calcium administration resulted in small (13-20%) reductions in iPTH, whereas some 4/5 (77-81%) of the nephrogenous cyclic AMP was rapidly and uniformly suppressed. In the patients with primary hyperparathyroidism, both analyses revealed a lack of absolute parathyroid autonomy in response to calcium, with overlapping iPTH responses between a majority of the patients and the control group. In contrast, the nephrogenous cyclic AMP responses provided a clear separation of the 2 groups after both calcium infusion and calcium injection (mean values for both studies, patients: 2.93 nmol/100 ml GF vs. normal sugjects: 0.38 nmol/100 ml GF), and measurements of total cyclic AMP excretion also clearly distinguished the 2 groups. When a sensitive antiserum with predominantly carboxy-terminal reactivity was employed, the iPTH responses to calcium injection provided an improved separation of patients and normal subjects. The data suggest that 1) although parathyroid autonomy is not, in general, a feature of primary hyperparathyroidism, abnormal parathyroid suppressibility is easily demonstrated even in patients with a subtle form of the disorder; 2) the determination of nephrogenous cyclic AMP provides an optimal method for assessing rapid changes in parathyroid function; and 3) the interpretation of iPTH results from such studies is dependent on a number of technological features of the assay employed.