The utility of preoperative parathyroid localization remains controversial. The gold standard for identification of abnormal parathyroid glands is exploration of the neck by an experienced surgeon. However, both for the experienced and less experienced surgeon, it may be desirable to localize the abnormality before surgery. Such a study would not only direct the surgeon to the site of the lesion but also would help reduce unnecessary dissection, the number of negative explorations, and operative anesthesia time. Unfortunately, currently used noninvasive techniques including ultrasonography, thallium-technetium subtraction, computed tomography scan, and magnetic resonance imaging have had only limited success (35% to 78%) in demonstrating abnormal parathyroid glands. Thirty patients with proven hyperparathyroidism were enrolled to compare a new imaging agent, technetium 99m sestamibi, as an alternative to thallium 201 for subtraction scintigraphy with technetium 99m pertechnetate. Only 14 of 30 patients operated on at George Washington University underwent an identical surgical approach (exploration of all four glands) and had complete preoperative calcium and parathormone levels, postoperative calcium levels, and total time of surgical procedures to formulate the basis of this report. The remaining 16 had their surgeries at other institutions. A surgical approach different from that used at George Washington University and unavailability of postoperative calcium levels and times of surgical procedures from these institutions necessitated deletion of these 16 patients from this report. Technetium 99m sestamibi-technetium 99m subtraction scans accurately localized the abnormal parathyroid gland in 13 of 14 patients, as compared with only 9 of 14 thallium 201-technetium 99m pertechnetate subtraction scans. These results support technetium 99m sestamibi scanning as the superior modality for preoperative parathyroid localization.