Objectives: To compare the results of magnetic resonance imaging (MRI) and thallium-201 (201Tl) SPET scanning with laboratory analyses including CSF DNA detection, brain biopsy, and necropsy in the discrimination of cerebral lymphoma and toxoplasmosis in patients with AIDS. Methods: A retrospective study of 32 patients infected with HIV who had focal CNS lesions on MR/as a result of either lymphoma or toxoplasmosis. Results: 18 patients had lymphoma, 12 had toxoplasmosis, and two had both. Toxoplasma IgG antibodies were detected in only seven patients - four with toxoplasmosis, two with lymphoma, and one with both diagnoses. Epstein-Barr virus DNA was detected in CSF of all six patients with lymphoma and none of two with toxoplasmosis. MRI showed multiple lesions in 23 patients, appearances did not discriminate between lymphoma and toxoplasmosis; nine patients had single lesions, of these eight had lymphoma (p=0.044, two tailed Fisher's exact test) 201Tl SPET showed accumulation in 17 with lymphoma and six with toxoplasmosis (p=0.034, two tailed Fisher's exact test). Of nine patients with single lesions on MRI and 201Tl SPET with focal accumulation eight had lymphoma. 201Tl SPET uptake ratios of ≤2.9 were only seen with lymphoma. Conclusion: Knowledge of patients' toxoplasma serostatus does not aid discrimination between lymphoma and toxoplasmosis. Single lesions on MRI with focal accumulation of 201Tl strongly suggest lymphoma. Multiple lesions on MRI with 201Tl SPET uptake ratios ≤2.9 also suggest lymphoma; uptake ratios less than 2.1 do not aid discrimination. Detection of Epstein- Barr virus DNA in CSF is highly sensitive and specific for cerebral lymphoma.