Technetium99m (99mTc)-Sestamibi/123I subtraction scanning was prospectively performed in 30 patients with primary hyperparathyroidism in an attempt to locate enlarged glands before first surgery. Imaging results were compared to surgical findings; the surgeon tried to identify all parathyroid glands. Twenty-seven patients were found to have a solitary adenoma during surgery, and 3 had multiglandular parathyroid hyperplasia. Twenty-six parathyroid adenomas (96%) were accurately located before surgery. The smallest gland detected weighted 125 mg. Preoperative detection of two mediastinal adenomas allowed them to be excised by median sternotomy during the initial operation. 99mTc-Sestamibi/123I subtraction scanning predicted multiglandular involvement in two patients with parathyroid hyperplasia, whereas it showed a solitary image in the third. Ten patients (33%) had associated nodular thyroid disease, hindering image analysis and leading to one false positive result. 99mTc-Sestamibi scanning seems to be better for locating enlarged parathyroid glands than other noninvasive imaging techniques. However, 1) difficulties associated with thyroid nodules call for complementary thyroid scanning; and 2) images showing a solitary enlarged parathyroid gland do not rule out multiglandular disease. This technique should help in detecting lesions, such as mediastinal glands, that are difficult to find at initial surgery.