Clinical samples from immunocompromised patients were screened for polyomaviral sequences by nested polymerase chain reaction (PCR) to evaluate the association of these viral infections with progressive multifocal leukoencephalopathy (PML). JC virus (JCV) DNA was detected in 19 of 23 CSF samples and all four brain samples from patients with PML. Neither BK virus (BKV) nor simian virus 40 (SV40) DNA were detected in these samples. No evidence was found to support the hypothesis that polyomaviral DNA is present in the central nervous system of immunosuppressed patients without PML (CSF n = 67, brain n = 19). JCV DNA was not detected in any peripheral blood sample included in this study. JCV DNA was detected in urine from three of eight patients with PML, but was also amplified from three of 29 urine samples from patients without PML. JCV, and not SV40 or BKV, was associated with PML in this study.