Using the technique of in situ DNA-to-DNA hybridization, a JC virus biotinylated DNA probe was developed and applied to formalin-fixed, paraffin-embedded, or fixed, frozen sections of brain tissue from three subjects with progressive multifocal leukoencephalopathy (PML). Light microscopy was carried out to correlate the presence of JC virus DNA with the selective infection of oligodendrocytes and astrocytes in PML. Oligodendrocytes (lytically infected) showed the greatest evidence of viral DNA. More astrocytes showing bizarre morphological changes had evidence of viral DNA than did astrocytes that were simply reactive. Viral DNA was not evident in vascular endothelial cells using this technique. Viral DNA replication may be an important initial step which produces the bizarre "transformed" astrocytes of PML. Findings in this study do not support the hypothesis that vascular endothelial replication is important in the pathogenesis of JC virus-induced PML. In situ hybridization with biotinylated JC virus probe may be useful in the diagnosis of PML on brain biopsy specimens.