The purpose of the present study was to assess the ability of technetium-99m-tetrofosmin (99mTc-TF) to predict tumor malignancy and to compare its uptake with that of thallium-201 (201Tl), technetium-99m-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) and fluorine-18-fluorodeoxyglucose (18F-FDG) in brain tumors. 99mTc-TF single-photon emission computed tomography (SPECT) imaging was performed in 22 patients with brain tumors and 3 healthy controls. Some of the patients underwent 201Tl (n = 12) and 99mTc-MIBI SPECT (n = 14) and 18F-FDG positron emission tomography (PET) (n = 12). The radioactivity ratio of tumor to contralateral normal tissue (T/N) and the ratio of tumor to contralateral white matter (T/WM) were calculated in SPECT and PET images, respectively. In healthy controls, 99mTc-TF uptake was seen only in scalp, in the choroid plexus and pituitary gland, but not in normal cerebral parenchyma. TF T/N in low grade gliomas (2.8+/-0.4) was significantly lower than that in high grade gliomas (22.5+/-29.8) and malignant non-gliomas (8.3+/-2.8) without overlap of values (p = 0.003 and p = 0.014, respectively). TF T/N was significantly correlated with MIBI T/N (p = 0.92, p = 0.001), Tl T/N (p = 0.72, p = 0.017), and FDG T/WM (p = 0.65. p = 0.031). There was an excellent agreement between TF T/N and MIBI T/N values on linear regression analysis (MIBI T/N = -0.63+/-0.97 x TF T/N). These preliminary results indicate that SPECT imaging with 99mTc-TF may be useful for the non-invasive grading of brain tumors. They also suggest that 99mTc-TF and 99mTc-MIBI may accumulate in brain tumors by a similar mechanism or in relation to a similar process of tumor cell proliferation.