The aim of this study was to evaluate the efficacy and role of technetium-99m tetrofosmin for the detection of abnormal parathyroid glands to be referred for surgical treatment. Twenty-eight consecutive patients, including 25 primary and 3 secondary cases of hyperparathyroidism, were evaluated. (99m)Tc-tetrofosmin/(99m)Tc-pertechnetate subtraction scintigraphy (TF/Tc) was performed on all patients, and the results were directly compared with those of (99m)Tc-methoxyisobutylisonitrile (MIBI)/(99m)Tc-pertechnetate subtraction scintigraphy (MIBI/Tc), (201)Tl/(99m)Tc-pertechnetate subtraction scintigraphy (Tl/Tc), magnetic resonance imaging (MRI) and ultrasonography (US). In cases of single-gland disease, the sensitivities of TF/Tc, MIBI/Tc, Tl/Tc, MRI and US were 63.2%, 68.4%, 57.9%, 55.6% and 63.2%, respectively. In cases of multi-gland disease, the sensitivities of TF/Tc, MIBI/Tc, Tl/Tc, MRI and US were 41.7%, 41.7%, 37.5%, 58.3% and 54.2%, respectively. In cases of parathyroid adenoma, the sensitivities of TF/Tc, MIBI/Tc, Tl/Tc, MRI and US were 68.8%, 75.0%, 68.8%, 62.5% and 75.0%, respectively. In cases of parathyroid hyperplasia, the sensitivities of TF/Tc, MIBI/Tc, Tl/Tc, MRI and US were 40.7%, 40.7%, 33.3%, 53.8% and 48.1%, respectively. It is concluded that, for the detection of abnormal parathyroid glands, (99m)Tc-tetrofosmin is as useful as (99m)Tc-MIBI and is more useful than (201)Tl.