Polymerase chain reaction (PCR) was used in the detection of JC virus (JCV) DNA in formalin-fixed, paraffin-embedded brain tissue sections from 24 patients with progressive multifocal leukoencephalopathy. Brain sections from normal autopsies (n = 17) and other neurologic conditions (n = 4) were used as controls. Specific amplified products were detected in 20 (83%) of 24 patients with progressive multifocal leukoencephalopathy. PCR did not amplify JCV or human beta-globin gene sequences in four patients with characteristic demyelinating lesions of progressive multifocal leukoencephalopathy that were positive for JCV by in situ hybridization or immunocytochemistry. PCR from biopsy sections resulted in more intense amplification signals than PCR from autopsy tissue. Normal brain sections from 17 autopsies were negative by PCR. Low-grade amplification of JCV was observed in one patient with herpes simplex virus encephalitis. PCR served as a more rapid test for confirmation of progressive multifocal leukoencephalopathy than in situ hybridization. However, PCR performance was altered by prolonged formalin fixation of tissue and undetermined inhibitors of the amplification reaction. Laboratories and clinicians should be aware of the difficulties encountered when using paraffin-embedded tissue for PCR.