BACKGROUND Sestamibi/iodine subtraction single photon emission computed tomography (SPECT) has been used successfully for the preoperative localization of adenomatous and hyperplastic parathyroid tissue in primary hyperparathyroidism, but the clinical usefulness of this technique in secondary hyperparathyroidism remains uncertain. The purpose of this study was to evaluate parathyroid localization that uses sestamibi/iodine subtraction SPECT in patients with secondary hyperparathyroidism before reoperative parathyroid surgery. METHODS Fourteen consecutive patients with chronic renal failure and secondary hyperparathyroidism who had previously undergone total parathyroidectomy combined with parathyroid autotransplantation in a sternocleidomastoid muscle were studied. Before reoperation, each patient received 400 microCi of sodium iodide I 123 orally and 20 to 25 mCi of technetium Tc 99m ((99m)Tc)-sestamibi intravenously, followed by sestamibi/iodine subtraction SPECT of the neck and chest. At surgery, the location, weight, and histopathologic results of all identified parathyroid tissue were recorded. RESULTS At surgery, 1 hyperplastic parathyroid gland was resected from each of 13 patients; including 1 undescended gland, 6 parathyroid autotransplants, and 5 mediastinal glands. The mean weight of the resected parathyroid glands was 1707 mg (range, 85-5300 mg). Sestamibi/iodine subtraction SPECT correctly identified and localized all 13 parathyroid glands (100% sensitivity) and was negative in the 1 patient whose surgery was unsuccessful. CONCLUSIONS The (99m)Tc-sestamibi/(123)I subtraction SPECT is able to correctly localize hyperplastic parathyroid tissue in patients with secondary hyperparathyroidism who have previously undergone parathyroid surgery and is a clinically useful study before reoperation.