Flow cytometric analysis of the nuclear deoxyribonucleic acid (DNA) content of parathyroid glands excised from patients with hypercalcemic hyperparathyroidism has identified three distinct DNA patterns. The most frequent pattern showed a high percentage of cells with tetraploid DNA, which indicated an increase in the G2 and M phase of the cell cycle. Thirty-four patients were found to have abnormal tetraploid DNA content. One patient had a normal diploid pattern, and seven were found to have an aneuploid DNA population in their excised parathyroid glands. This unexpected finding of aneuploid DNA appears to be an unique feature of these endocrine glands because they have no histologic or clinical characteristics of malignant change. All patients have remained normocalcemic and clinically well after excision of only grossly enlarged glands. Postoperative parathyroid hormone (PTH) levels were correlated in 17 patients with DNA analyses of biopsy specimens from 30 normal-sized glands which were left in situ. Seven patients with elevated PTH postoperatively had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had high tetraploid or aneuploid DNA in all 13 glands from which biopsy specimens had been taken. In 10 patients with normal PTH levels, six had normal diploid patterns, whereas four had high tetraploid DNA in their gland biopsy specimens. DNA content present in biopsy specimens of normal-sized, in situ glands was predictive (p less than 0.042) of parathyroid gland secretory activity. These findings suggest that the stimulus for parathyroid gland hyperfunction often affects more than a single enlarged gland and persists after clinical cure, as shown by a more rapid cell turnover in some remaining glands and continued hypersecretion of hormone.