JC virus (JCV) is a common human polyomavirus that infects 70-80% of the population worldwide. In immunosuppressed individuals, JCV infects oligodendrocytes and causes a fatal demyelinating disease known as progressive multifocal leukoencephalopathy (PML). The tropism of JCV is restricted to oligodendrocytes, astrocytes, and B lymphocytes. Several mechanisms may contribute to the restricted tropism of JCV, including the presence or absence of cell-type-specific transcription and replication factors and the presence or absence of cell-type-specific receptors. We have established a system to investigate cellular factors that influence viral tropism by selecting JCV-resistant cells from a susceptible glial cell line (SVG-A). SVG-A cells were subjected to several rounds of viral infection using JC virus (M1/SVE Delta). A population of resistant cells emerged (SVGR2) that were refractory to infection with the Mad-4 strain of JCV, the hybrid virus M1/SVE Delta, as well as to the related polyomavirus SV40. SVGR2 cells were as susceptible as the SVG-A cells to infection with an unrelated amphotropic retrovirus. The stage at which these cells are resistant to infection was investigated and the block appears to be at early viral gene transcription. This system should ultimately allow us to identify glial specific factors that influence the tropism of JCV.