To evaluate the diagnostic value of thallium-201 single-photon emission computed tomography (201Tl SPECT) in the management of focal brain disorders in the era of highly active antiretroviral therapy (HAART), a validation study of diagnostic procedure was performed in a tertiary clinical care center in Italy. Thirty-eight consecutive HIV-infected patients with neurological impairment and focal brain lesions (FBL) were enrolled in a prospective evaluation and underwent diagnostic procedures according to a standardized protocol based on modified previously released guidelines. Six out of seven PCNSL presented high uptake at 201Tl SPECT [sensitivity 86% (95% CI 42-99); specificity 77% (95% CI 58-90); positive predictive value (PPV) 46% (95% CI 20-74); negative predictive value (NPV) 96% (95% CI 78-100)]. Among toxoplasmic encephalitis (TE) cases 14 showed no uptake and 5 showed an increased uptake [sensitivity 74% (95% CI 49-90); specificity 42% (95% CI 21-66); PPV 56% (95% CI 35-75); NPV 61% (95% CI 32-85)]. Patients taking HAART were more likely to display an increased uptake of 201Tl in the cerebral lesions than patients without HAART (OR 5.07; 95% CI 1.19-21.5). Considering only the patients with diagnosis of TE, 60% of patients who showed high radionuclide uptake were taking HAART, while 79% of patients without relevant uptake were not taking HAART. As a result of the impact of HAART, the diagnostic value of 201Tl SPECT in the management of HIV-associated FBL could be substantially reduced. This observation suggests that in HAART-treated patients, this diagnostic tool be used only when combined with other more specific diagnostic markers.