At present it remains unknown if the JC virus (JCV), the causative agent of progressive multifocal leukoencephalopathy, employs specific cellular receptors or cell membrane factors for viral entry. To investigate this, me have examined the cell-specific replication of a chimeric JCV, which contains the control region (CR) cloned from JCI and the coding sequences of the prototype Mad-1 virus. We examined and compared the hemagglutination (HA) titers produced during viral multiplication following microinjection, transfection with chloramphenicol acetyltransferase (CAT) assays, and inoculation using a permissive cell line IMR-32 and three non-permissive cell lines COS-7, CV-1, and A431. Virus microinjected into cells proliferated well in both IMR-32 and COS-7, but not in the non-permissive CV-1 and A431 cell lines. When cells were infected with the chimeric JCV, the HA titers were high in IMR-32, low in COS-7, and negative in the other cell lines. Chloramphenicol acetyltransferase assays demonstrated that the CR was active in IMR-32, COS-7, and CV-1, and inactive in A431 cells, The results suggest that not only nuclear, but also cell membrane factors play an important role in the susceptibility of cells to JCV infection.