The aims of this study were to assess the clinical usefulness of [Tc-99m] tetrofosmin (TF) single photon emission computed tomography (SPECT) and X-ray transmission computed tomography (CT), performed simultaneously with a hybrid imaging device for the functional anatomical mapping of brain tumors and to evaluate the additional information of SPECT/CT when compared to SPECT alone. Thirty (30) patients were studied: 20 were evaluated before undergoing surgery and 10 after surgery and before radiotherapy planning. The acquisition of both functional (SPECT) and morphologic (CT) images were obtained in a single session. SPECT images were firstly evaluated alone and then reinterpreted by adding the anatomical (CT) planes. Fusion imaging was successfully obtained in all patients with precise correspondence between SPECT and CT slices. SPECT/CT had a significant clinical impact in 13 (43.3%) of 30 cases; in particular, SPECT/CT accurately characterized eight lesions near sites of physiological uptake (i.e., four near ventricles/choroids plexus, three near venous sinuses, one near the skull) and localized viable tumor tissue in 5 patients evaluated after surgery. SPECT/CT with TF using this hybrid device represents a useful clinical tool in brain tumor imaging, both correctly categorizing focal areas near sites of physiological uptake and localizing viable tumor tissue after surgery.