Isolates of the human polyomavirus JC can be grouped as either PML-type or archetype strains primarily on the basis of divergence in their regulatory regions. Only PML-type viruses have so far been found to be associated with the human demyelinating disease progressive multifocal leukoencephalopathy. Here we have compared the functional properties of archetype and PML-type regulatory regions with regard to DNA replication and viral gene expression. No significant differences could be detected between archetype and PML-type regions in their ability to direct episomal DNA replication in the presence of JC virus T antigen. When viral gene expression was examined, early- and late-gene promoters from all PML-type strains exhibited a significantly higher activity in glial than in nonglial cells. Surprisingly, archetype strain promoters were also preferentially active in glial cells, although this effect was less pronounced than in PML-type strains. Furthermore, all promoters from archetype strains reacted to the presence of viral T antigen or the glial transcription factor Tst-1/Oct6 in a manner similar to the promoters of the PML-type viral strain Mad-1. Interestingly, T antigen and Tst-1/Oct6 were found to function in a species-specific and cell-type-specific manner, respectively. We concluded from our experiments that the differences in the regulatory regions cannot account for the different biology of archetype and PML-type viral strains.