The purpose of this study was to evaluate the usefulness of 99Tcm-MIBI accumulation for the differentiation of histological diagnosis of malignant brain tumours in comparison with the findings obtained using 201Tl chloride. A total of 25 patients with malignant brain tumours were investigated. The histological categories of tumours included glioblastoma multiforme (n = 5), anaplastic astrocytoma (n = 4), malignant lymphoma (n = 5), and metastatic tumour (n = 11). Simultaneous dual single photon emission tomography (SPET) images with 99Tcm-MIBI and 201Tl were acquired 15 min (early) and 2 h (delayed) after injection, and the early ratio, delayed ratio and retention index were measured. The new indices 201Tl/99Tcm-MIBI ratios and 201Tl/99Tcm-MIBI retention index were also calculated. With respect to the histological type, a higher retention index using 99Tcm-MIBI was noted in glioblastoma multiforme compared with metastatic tumour. Higher values of both ratios using 201Tl were noted in glioblastoma multiforme compared to metastatic tumour. The value of the delayed ratio obtained using 201Tl was higher in glioblastoma multiforme than in anaplastic astrocytoma, and the value was also higher in malignant lymphoma than in metastatic tumour. The 201Tl/99Tcm-MIBI early ratio of glioblastoma multiforme was significantly higher than that of metastatic brain tumour. The 201Tl/99Tcm-MIBI retention index of malignant lymphoma was significantly higher than that of glioblastoma multiforme. In the histological type of tumour, 99Tcm-MIBI is not superior to 201Tl, but the combined indices using 201Tl/99Tcm-MIBI may add new information about differential diagnosis.