The accurate identification of the location of hyperfunctioning parathyroid nodules is essential to the effective surgical treatment of hyperparathyroidism. Radionuclide imaging has played an important role in the detection of pathological parathyroid nodules. Sestamibi is a new radiopharmaceutical with a good affinity for parathyroid tissue that images hyperfunctioning parathyroid lesions as hot nodules. The double-phase protocol with early (ten minutes) and delayed (120 minutes) imaging is effective fordetecting the parathyroid lesions. The use of a pinhole collimator improves the sensitivity, but attention should be paid to the ectopic gland. Most lesions heavier than 250 mg could be detected with this scintigraphy, but the detectability of hyperplastic glands is inferior to that of adenomas. A sestamibi scan is better for the detection of ectopic lesions and recurrent disease than ultrasonography, and its detectability is also superior to other radionuclide tests such as tetrofosimin scanning and the subtraction technique using thallium-201 and Tc-99m pertechnetate images. The application of sestamibi to radionuclide-guided parathyroidectomy has been studied with encouraging results. Sestamibi can also be used for the evaluation of autotransplanted glands.