Surgery for primary hyperparathyroidism is successful in 95% of patients, but ectopic glands and anatomic variations in location are causes of surgical failure. The radionuclide imaging agent, technetium (Tc)-99m-sestamibi, in conjunction with subtraction iodine-123 scanning, is a new method of preoperative localization of abnormal parathyroid glands. In a study approved by the Institutional Review Board, 22 patients with primary hyperparathyroidism underwent preoperative evaluation with high-resolution ultrasonography and Tc-99m-sestamibi/I-123 radionuclide scanning for attempted localization of abnormal parathyroid glands. Results of Tc-99m-sestamibi scanning and ultrasound were correlated with surgical and pathologic findings. Of 22 patients, 16 had a solitary parathyroid adenoma, 1 had a double adenoma, and 5 had diffuse parathyroid hyperplasia. The Tc-99m-sestamibi/I-123 radionuclide scan preoperatively identified a solitary adenoma in 14 of 16 patients (sensitivity: 88%). However, when the data were analyzed retrospectively along with surgical and pathologic findings, the Tc-99m-sestamibi scan correctly localized all parathyroid adenomas for a sensitivity of 100%. The one patient with a double adenoma had a localization image consistent with two enlarged glands. All patients with diffuse parathyroid hyperplasia had Tc-99m-sestamibi imaging consistent with diffuse hyperplasia, although delineation of individual enlarged glands was not possible. High-resolution ultrasound identified 11 of 16 parathyroid adenomas (sensitivity: 69%). The patient with a double adenoma had a negative ultrasound. Ultrasound was less accurate in five patients with diffuse hyperplasia: one scan was completely negative, two scans revealed only one enlarged gland, and two scans revealed two enlarged glands. The Tc-99m-sestamibi/I-123 subtraction radionuclide scan is more sensitive than high-resolution ultrasonography for the preoperative localization of abnormal parathyroid glands. Tc-99m-sestamibi/I-123 radionuclide scanning may be more useful than ultrasonography to the surgeon in the preoperative localization of abnormal parathyroid glands.