In this multicenter, retrospective study of 160 brain biopsies in the assessment of HIV-related focal brain lesions, diagnostic sensitivity was acceptable (87%), but the procedure carried considerable morbidity (7.5%) and mortality (3.1%). Moreover, it is not always possible to initiate the changes in therapy indicated by the results, and overall survival remains poor, with a median of 2 months. Criteria for brain biopsy for the diagnosis of focal brain lesions should be redefined to include selected patients for whom a less invasive approach does not yield a definitive diagnosis.