Primary and secondary cell cultures of brain tissue from two fatal cases of progressive multifocal leukoencephalopathy (PML) were closely monitored by light and electron microscopy and immunochemical labelling for glial fibrillary acidic protein. Although the cell cultures appeared heterogeneous and sometimes included fibroblasts, most of the cells were glial and many were identified as astrocytes by their morphology and immunochemical label. In long-term primary cultures four to four and a half months old and subcultures after many passages, focal cytopathic effect was noted in many cells. At the same time, papovavirions appeared in the nuclei of degenerating cells. However, the identity of these cells could not be established. Also, consistently present in the cultures were large atypical and pleomorphic cells that were likewise morphologically and immunochemically identified as astrocytes. Complete formed virions consistent with papovavirus appeared in the nuclei of some of these cells after several cell passages. The emergence of the virus in these cultured astrocytes supports the recently held notion that there may be in PML an abortive, or nonpermissive, papovavirus infection of this cell type which is believed to undergo cell transformation.