Central Nervous System (CNS) diseases that occur in HIV-positive patients are mainly due to HIV itself or to opportunistic microorganisms. Polyomavirus JCV, BKV and SV40 have been associated with encephalopathies in both HIV-positive and HIV-negative patients. To investigate the presence of Polyomavirus DNA sequences in patients affected by CNS disorders, 82 CSF samples from 70 HIV-positive and 12 HIV-negative patients were analyzed by PCR. A double HIV and SV40 infection was found in one patient suffering with AIDS dementia complex. SV40 DNA sequence analysis showed the homology with wild type SV40 strain. SV40 should be considered as a potential causal agent of CNS disorders in AIDS patients.