Cerebrospinal fluid (CSF) from 12 AIDS patients with clinical signs consistent with progressive multifocal leukoencephalopathy (PML) was examined by the polymerase chain reaction (PCR) for the presence of JC virus (JCV). A specific JCV target sequence was amplified in the CSF from 9 of the 12 patients and also in brain tissue from all nine JCV-positive patients. The clinical course, neuroimaging features, and, in four cases, histopathology of brain tissue proved that the nine patients had PML. In the other three patients with central nervous system disorders, the JCV genome was undetectable by PCR and Southern blot analysis in CSF and brain tissue. The clinical course and neuroimaging features ruled out PML in these three patients. Five CSF samples and five brain tissue specimens from 10 PML-free AIDS patients with central nervous system disorders were all negative for the JCV genome by PCR and Southern blot analysis. These results show that detection of JCV in CSF by PCR is a good alternative to brain tissue studies for the virological diagnosis of PML in AIDS patients.