To evaluate the utility of technetium-99m (Tc-99m) sestamibi for visualization of functioning parathyroid tissue, 14 subjects underwent Tc-99m sestamibi 123I subtraction scanning as part of the preoperative evaluation for hyperparathyroidism. Informative scans were obtained in 13 subjects, including 7 patients with recurrent or persistent hyperparathyroidism, and correctly identified the location of the hyperfunctioning parathyroid tissue found at surgery. In all informative patients, hyperparathyroidism was due to adenomatous disease or hyperplasia secondary to renal failure. Successful scans were obtained with glands as small as 220 mg. In the lone patient in whom Tc-99m sestamibi scanning failed to localize hyperfunctioning parathyroid tissue, surgery revealed a 1700-mg hyperplastic parathyroid neoplasm in the neck. In no case did a Tc-99m sestamibi scan suggest parathyroid tissue where there was none. In 1 case, a patient presented with persistent hyperparathyroidism after 1 neck and a second combined neck and mediastinal exploration. Tc-99m sestamibi imaging revealed uptake in the periaortic region, and a 570-mg adenoma was found in the aortopulmonary window. Using only initial studies, prospective evaluation provided a sensitivity of 78.5% and a positive predictive value of 100%. After repeat studies in 5 patients, 2 of 3 patients with initially negative results and technically deficient scans became positive on restudy. Inclusion of these studies increased sensitivity to 93%. Tc-99m sestamibi 123I subtraction scanning appears to be a reliable noninvasive method for preoperative localization of hyperfunctioning parathyroid tissue.