Biotinylated DNA:DNA in situ hybridization was used for determining the presence of JC virus in brain tissues of 67 patients thought to have progressive multifocal leukoencephalopathy. Sixty patients had acquired immunodeficiency syndrome (AIDS) or other illnesses that decreased the cell-mediated immunity. Two patients had no underlying disease, and five others had chronic illnesses not typically associated with reduced cell-mediated immunity. In situ hybridization with biotinylated probe provides specificity and ease of interpretation. The presence of virus can be correlated at the single-cell level with attendant pathologic changes in oligodendrocytes and astrocytes. Not only archival tissue but also tiny fragments of brain biopsy material can be evaluated successfully. Quantifying the technique suggests that the nucleus of a cell labeling for JC virus DNA averages 1,000 copies of replicating genome. Identification of an infected cell is pathologically significant even when only a few such cells are present in a biopsy specimen. Biotinylated DNA or RNA probes are equally effective in identifying infected cells. In situ hybridization will likely continue to be a useful adjunctive procedure for the evaluation of brain tissue from patients suspected of having progressive multifocal leukoencephalopathy.