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Cerebrospinal fluid (CSF) samples from 49 acquired immunodefficiency disease syndrome (AIDS) patients with a central nervous system (CNS) disease were examined by polymerase chain reaction (PCR) to evaluate the association between the positivity for cytomegalovirus (CMV) and Epstein-Barr virus (EBV), and clinical diagnosis of a CNS disease. Frequency and clinical relevance of detection of DNA of human herpesviruses 6 (HHV-6), 7 (HHV-7) and 8 (HHV-8) were also determined. DNA of one or more of the following viruses was found in 26 of 49 patients (53%): CMV in 16 (33%), EBV in 13 (27%), human herpesvirus 6 (HHV-6) in 2 (4%), human herpesvirus 7 (HHV-7) in 1 (2%), and human herpesvirus 8 (HHV-8) in 1 (2%). The CMV detection was significantly associated with encephalitis and peripheral neuropathy (7/16 vs. 2/33, p = 0.003), while EBV with primary CNS lymphoma (P-CNSL) (8/13 vs. 0/36, p < 0.0001). HHV-6 DNA was found in CSF of two patients with neuroradiological features suggestive of cerebral lesions. HHV-8 or HHV-7 DNA was detected in the CSF of patients with unexplained neurological symptoms. This study confirms that the PCR analysis of CSF is a valid tool for the diagnosis of neurological diseases associated with CMV and EBV. On the other hand, HHV-6, HHV-7 and HHV-8, instead, were rarely detected in CSF of AIDS patients and have certainly no correlation with the CNS disease found.