Microglial nodules associated with opportunistic and HIV-related lesions are frequently found in the brains of AIDS patients. However, in many cases, the causative agent is only presumptively suspected. We reviewed 199 brains of AIDS patients with micronodular lesions to clarify their etiology by immunohistochemistry (to Toxoplasma gondii, cytomegalovirus, herpes simplex virus I/II, varicella zoster virus and HIV-p24 core protein), PCR (for herpetic viruses and Mycobacterium tuberculosis) and electron microscopy. Productive HIV infection was observed in 110 cases (55.1%): 30 cases with Toxoplasma gondii encephalitis, 30 with cytomegalovirus encephalitis, eight with multiple cerebral diseases, while in the remaining 42 cases HIV was the only pathogenetic agent. Multinucleated giant cells (hallmark of HIV infection) were found in the MGNs of 85/110 cases with HIV-related lesions; the remaining 25 cases had only p24 positive cells but no multinucleated giant cells. In these latter cases the micronodular lesions had been initially attributed to the main opportunistic agent found in the brain, or defined as subacute encephalitis. Individual microglial nodules positive for an opportunistic pathogen were generally negative for HIV antigens. In 13 cases no opportunistic agent or HIV productive infection was found. In these cases, PCR and electron microscopy examination for HIV and other viral infections were negative. Our data suggest that HIV-immunohistochemistry should be used for the etiological diagnosis of micronodular lesions in AIDS brains, even in the presence of other pathogens. After extensive search, the etiology of the microglial nodules remains unknown in only a small percentage of cases.