Tc-99m sestamibi parathyroid imaging was performed in 28 patients with chronic renal failure to localize abnormal parathyroid glands in patients receiving hemodialysis, and compared the localization with ultrasonography and magnetic resonance (MR) imaging. Methods: We imaged 28 patients with secondary hyperparathyroidism using Tc-99m sestamibi (about 600 MBq) at 10 min and 2–3 h following radiotracer injection. In addition, mediastinal images were recorded at approximately 1 h following injection to identify ectopic parathyroid glands. All patients also were evaluated with ultrasonography and MR imaging. Results: Tc-99m sestamibi scans demonstrated focal uptake in 60 glands of the 28 patients, and was categorized as slight uptake in 71.7% (43/60), and intense uptake in 28.3% (17/60). Seventeen of the 28 patients underwent parathyroidectomy. A total of 64 glands were resected. Sestamibi imaging was more sensitive for localizing abnormal parathyroid glands than ultrasonography or MR imaging. Histologic evaluation of 27 resected parathyroid glands revealed that the number of oxyphil or chief cells was not proportional to sestamibi uptake. Conclusion: Our data indicate that Tc-99m sestamibi imaging should be used initially to localize abnormal parathyroid glands in hemodialysis patients with secondary hyperparathyroidism, prior to MR imaging or ultrasonography. Sestamibi uptake in parathyroid glands may not correlate with the degree of hypercellularity of oxyphil cells.