Progressive multifocal leukoencephalopathy is a subacute demyelinating disease of the central nervous system (CNS) resulting from opportunistic infections in immunocompromized patients infected with a common polyomavirus, JC virus (JCV). Unlike other polyomaviruses, JCV exhibits an unusually narrow tissue tropism by primarily infecting glial cells of the CNS. JCV DNA replication is similar to that of the well-characterized papovavirus, SV40, which requires the viral early protein T-antigen and host-replication factors including DNA polymerases and DNA topoisomerase I. In this study we have been able to effectively block replication of viral DNA in glial cells using camptothecin, a drug which inhibits DNA topoisomerase activity. Pulse-treatment of cells with non-toxic levels of camptothecin specifically blocks viral DNA replication with no inhibitory effect on host transcription and translation processes as examined by viral gene expression in the transfected cells. Furthermore, drug treatment of the cells exhibits no significant effect on DNA topoisomerase I gene transcription. We further demonstrate that repeated pulse-treatment of cells with the drug is required for complete blockage of viral DNA replication. The importance of these findings in the treatment of AIDS encephalopathy is discussed.