IC virus (ICY) has frequently been described as a tissue specific agent in the human population targeting the myelin producing oligodendrocyte whose destruction results in the demyelinating disease, progressive multifocal leukoencephalopathy (PML). IC virus growth in cell culture systems has generally reflected that observation because its multiplication is most efficient in cultures derived from human fetal brain. These cultures, however, are composed of a mixed population of cells phenotypically categorized as either astrocytes or precursors to oligodendrocytes. We have been able to separate mixed populations of glial cells from human fetal brain in culture and produce a pure population of astrocytes. We then analyzed the astrocyte cell cultures for their ability to support ICY transcription, replication, and virion production. Our results demonstrate that cultures of astrocytic cell types support JCV gene expression leading to virus multiplication. These astrocytic cell cultures also can be passaged several times in culture without loss of their astrocytic phenotypes or susceptibility to JCV infection. These data are consistent with our studies of JCV gene expression in fixed brain tissues of PML patients and virus immortalized astrocytic cell lines. These results strongly suggest that astrocytes as well as oligodendrocytes playa role in the pathogenesis of PML and should focus additional experiments on this particular cell type.