The aim of this study was to evaluate the efficacy of 99mTc-MIBI and 123I subtraction scintigraphy for the detection of abnormal parathyroid glands to be referred for surgical treatment. Thirty-nine consecutive patients, including 35 primary and four secondary cases of hyperparathyroidism, were evaluated. 99mTc-MIBI/123I subtraction scintigraphy (MIBI/I) was performed on all patients, and the results were compared with delayed images of 99mTc-MIBI (D-MIBI), magnetic resonance imaging (MRI) and ultrasonography (US). The overall sensitivity of MIBI/I, MRI, US and D-MIBI was 55.9%, 43.4%, 50.8% and 39.0%, respectively. In cases of single-gland disease, the sensitivity of MIBI/I, MRI, US and D-MIBI was 62.1%, 48.3%, 55.2% and 44.8%, respectively. In cases of multi-gland disease, the sensitivity of MIBI/I, MRI, US and D-MIBI was 50.0%, 37.5%, 46.7% and 36.7%, respectively. In cases of parathyroid adenoma, the sensitivity of MIBI/I, MRI, US and D-MIBI was 71.4%, 50.0%, 71.4% and 50.0%, respectively. In cases of parathyroid hyperplasia, the sensitivity of MIBI/I, MRI, US and D-MIBI was 55.2%, 42.3%, 50.0% and 39.7%, respectively. It is concluded that 99mTc-MIBI/123I subtraction is more useful than the delayed imaging of 99mTc-MIBI, MRI and US.