Initiation of polyomavirus DNA replication in eukaryotic cells requires the participation of the viral early protein T antigen, cellular replication factors, and DNA polymerases. The human polyomavirus JC virus (JCV) is the etiologic agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy in immunocompromised individuals. This virus exhibits a narrow host range and a tissue specificity that restricts its replication to glial cells of the central nervous system. Restriction of viral DNA replication due to species specificity of the DNA polymerase, coupled with glial cell-specific transcription of the viral early promoter, is thought to account for the brain-specific replication of JCV. In this report we demonstrate that overexpression of Pur alpha, a protein which binds to single-stranded DNA in a sequence-specific manner, suppresses replication of JCV DNA in glial cells. Results from footprinting studies indicate that Pur alpha and T antigen share a common binding region spanning the single-stranded ori sequence of JCV. Further, T antigen was capable of stimulating the association of Pur alpha with the ori sequence in a band shift assay. Whereas no evidence for simultaneous binding of Pur alpha and T antigen to single-stranded DNA has been observed, results from coimmunoprecipitation and Western blot (immunoblot) analyses of proteins derived from cells producing JCV T antigen indicate a molecular association of JCV T antigen and Pur alpha. The binding of Pur alpha to the single-stranded ori sequence and its association with T antigen suggest that Pur alpha interferes with the activity of T antigen and/or other regulatory proteins to exert its negative effect on JCV DNA replication. The importance of these findings in the reactivation of JCV in the latently infected individual under immunosuppressed conditions is discussed.