Neurological diseases and a variety of neoplasms frequently occur in AIDS patients. Human JC and BK polyomaviruses have been associated with neurological disorders in such patients. SV40 polyomavirus sequences have been detected in human brain tumours, other neoplasms and normal tissues. JCV, BKV and SV40 DNA sequences were investigated in cerebrospinal fluid (CSF) samples from 12 AIDS patients affected by different neurological disorders, by PCR assay and filter hybridisation with specific internal oligoprobes, and DNA sequencing. Three of the 12 CSF samples were positive for JCV (one sample) or SV40 (one) DNA, or both (one). No sample was positive for BKV DNA. JCV- and SV40-specific genomic regions were confirmed by DNA sequencing. CSF samples from the two patients diagnosed clinically as having progressive multifocal leukoencephalopathy (PML) contained either JCV (one sample) or SV40 (one) DNA. The CSF found to contain both JCV and SV40 DNA originated from a patient with a cerebral mass lesion of unknown aetiology. These results suggest that SV40 may be involved in the aetiology of PML in AIDS patients, and raise the possibility that SV40 and JCV may act synergically in vivo to enhance their pathogenicity.