BACKGROUND Until now, preoperative parathyroid imaging has been considered unnecessary because currently available techniques do not provide any better results than an expert surgeon. We conducted a multicenter prospective study evaluating the capability of technetium 99m sestamibi scintigraphy in the preoperative identification of pathologic glands. METHODS Ninety-three patients with hyperparathyroidism, seven of them reoperative cases, were studied. In addition, 20 age-matched normocalcemic control subjects were also studied. RESULTS Surgical confirmation of scintigraphic images was obtained in 91 of 93 cases (sensitivity, 97.8%). In all reoperative cases (n = 7), in all cases of ectopic glands (whether operated or not; n = 6), and in all patients with a single-gland disease (n = 70), topographic identification of the pathologic gland was correct in 100%. In multiple-gland disease (n = 23), involvement of more than one gland was visualized in only 61% of the patients; however, in 91% of patients, at least one gland was localized. Surgical success-defined as postoperative normocalcemia-with this approach was 100%. All scans of normocalcemic control subjects were negative. Of 31 patients in whom a multinodular goiter coexisted, seven presented a significant radionuclide background at 120 minutes' scan. False-positive images were found together with those corresponding to the pathologic glands in only three cases. CONCLUSIONS 99mTc-sestamibi is a highly reliable, sensitive, and specific technique for imaging of pathologic glands in hyperparathyroidism, especially in single-gland disease. It may be considered as a first line single-procedure when a preoperative topographic diagnosis is required.