The aim of this study was to compare 201Tl-chloride and 99Tc(m)-sestamibi (MIBI) SPET brain imaging for differentiating brain lymphoma from other intracranial lesions in AIDS patients. Both studies were performed on the same day in 17 AIDS patients with intracranial enhancing lesions on either CT or MRI. Eleven patients underwent brain biopsy and six patients were followed clinically. We calculated the radiopharmaceutical uptake ratio of the lesion to that on the contralateral side with the guidance of CT or MRI findings. Ratios of 1.5 or more were considered to represent malignant lesions and ratios < 1.5 were considered to represent benign lesions. Biopsy revealed four cases of lymphoma, four cases of toxoplasmosis and two cases of progressive multi-focal leukoencephalopathy; one biopsy yielded necrosis. Both the MIBI and 201Tl studies yielded no false-negative cases of lymphoma (sensitivity 100%). Of the 13 non-lymphoma cases, the 201Tl studies showed seven true-negative cases (specificity 54%) and the MIBI studies showed nine true-negative cases (specificity 69%). The biopsies of the false-positive cases (toxoplasmosis) showed a pattern of healing after medical treatment. We conclude that MIBI is more helpful than 201Tl because of higher specificity and equal sensitivity. The medical treatment of toxoplasmosis is a cause of false-positive 201Tl and MIBI studies.