A procedure for rapid detection of JC virus (JCV) using the polymerase chain reaction is described. The procedure was tested in eight HIV-1-seropositive patients with progressive multifocal leukoencephalopathy. One-step DNA extraction using a chelating resin was carried out on clinical samples of cerebrospinal fluid (CSF), urine and brain tissue. After amplification, PCR products were detected by a DNA hybridization method. Microplates were coated with a specific probe and hybridized PCR products were revealed by a commercial colorimetric immunoassay. Using this procedure JC virus DNA was detected in all CSF specimens from patients with progressive multifocal leukoencephalopathy. This sensitive and rapid (24 h) procedure could greatly facilitate use of the DNA probe assay for detection of JC virus in clinical laboratories.