Our aim was to evaluate the use of dual-isotope thallium-201 (Tl) and technetium-99m sestamibi (sestamibi) simultaneous acquisition in brain single-photon emission tomography (SPET) for the differentiation between brain lymphoma and benign central nervous system (CNS) lesions in AIDS patients. Thirty-six consecutive patients with enhancing mass lesions on magnetic resonance (MR) imaging were included in the study. SPET of the brain was performed to obtain simultaneous Tl and sestamibi images. Regions-of-interest were drawn around the lesion and on the contralateral side to calculate uptake ratios. The final diagnosis was reached by pathologic findings in 17 patients and clinical and/or MR follow-up in 19 patients. Of the 36 patients, 11 had brain lymphoma, 1 glioblastoma multiforme, 15 toxoplasmosis and 9 other benign CNS lesions. Correlation between SPET and the final diagnosis revealed in 10 true-positive, 23 true-negative, 1 false-positive and 2 false-negative studies. All patients with toxoplasmosis had negative scans. A patient with a purulent infection had positive scans. Tl and sestamibi scans were concordant in every lesion. The same lesions that took up Tl were also visualized with sestamibi. However, sestamibi scans showed higher lesion-to-normal tissue uptake ratios (3.7 +/- 1.8) compared with those of Tl (2.3 +/- 0.8, P < 0.002). Simultaneous acquisition of Tl and sestamibi can help differentiate CNS lymphoma from benign brain lesions in AIDS patients.