123I/99mTc-sestamibi subtraction single photon emission computed tomography (SPECT) has been proposed to detect hyperplastic parathyroid tissue, but the clinical usefulness of this technique in secondary hyperparathyroidism is uncertain. The purpose of this study was to evaluate preoperative parathyroid localization using 123I/99mTc-sestamibi subtraction SPECT in patients with renal failure and secondary hyperparathyroidism. Nineteen patients with chronic renal failure and secondary hyperparathyroidism underwent 123I/99mTc-sestamibi subtraction SPECT imaging preoperatively. None of these patients had undergone previous neck surgery. The location, weight, and histopathological results of all identified parathyroid glands were recorded. Surgery was considered successful in all patients, with resection of a total of 74 hyperplastic parathyroid glands. 123I/99mTc-sestamibi subtraction SPECT correctly identified 57 of these parathyroid glands (77% sensitivity). The mean weight among the true positive glands (n = 57) was 1031 mg (range, 45-7900 mg), and that among the false negative glands (n = 17) was 465 mg (range, 20-1800 mg). This difference between the mean weights was statistically significant (P = 0.018). There was a positive correlation between parathyroid weight and detectability with 123I/99mTc-sestamibi subtraction SPECT (Spearman correlation = 0.28; P = 0.0167). 123I/99mTc-sestamibi subtraction SPECT is able to correctly localize hyperplastic parathyroid glands in patients with renal failure and secondary hyperparathyroidism, but there is a fairly weak relationship between preoperative detection rate and anatomical parathyroid gland size.