Progressive multifocal leukoencephalopathy (PML), caused by the human polyomavirus JC (JCV), is an opportunistic infection of the central nervous system (CNS), the histopathological diagnosis of which can be made by routine staining. Very low copy numbers of JCV nucleic acid can be detected in paraffin sections by the specific and highly sensitive in situ polymerase chain reaction (in situ PCR). The authors evaluated JCV infection in 12 acquired immunodeficiency syndrome (AIDS) patients with PML by comparison of hematoxylin and eosin (H&E) staining, in situ hybridization (ISH), and in situ PCR. Phenotype of infected cells was determined by immunohistochemistry with antibodies against glial fibrillary acidic protein (GFAP) or cluster of differentiation 68 (CD68), focusing on cells containing low JC viral copy numbers, and on cell types that are normally not associated with papovavirus infection. The number of detectable JCV-positive oligodendrocytes increased markedly upon PCR amplification and hitherto unknown oligodendrocytic staining patterns were discernible: JCV DNA was detectable in both nucleus and cytoplasm, in cytoplasm only, and as ghost-cell silhouettes appearing as a membranous "rim" of staining product in some cells. The authors suggest that the staining patterns correspond to different stages of the viral replication cycle. Some human immunodeficiency virus (HIV)-type giant cells (HIV-GCs) were shown to contain JCV DNA, thus probably revealing a double infection. Macrophages and HIV-GCs showed staining in the cytoplasm and the nuclei, indicating that they not only may phagocytize JCV particles but may also be actively infected. CD68-positive GCs were occasionally noted to contain a complete JCV DNA-positive nucleus in their center, and were accordingly called JCV-type giant cells (JCV-GCs). Rarely, JCV DNA signals were noted in vascular endothelium. No JCV infection was detectable in lymphocytes, neurons, or in brain tissue of JCV-negative age-matched controls. The authors report new findings concerning inter- and intracellular JCV infection patterns in PML, possibly shedding new light on JCV susceptibility of different cell types in the brain of AIDS patients with PML.