In the circular DNA genome of the human polyomavirus BK an approximately 400 bp non-coding control region (NCCR) separates the early and late genes. The NCCR contains the origin of replication as well as the promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription. The NCCR has been shown to be very heterogeneous between different BK virus (BKV) strains. This may affect the host cell permissivity and oncogenic potential of a given BKV strain. Our previous studies with BKT-1B, a continuous cell line established from a BKV (Gardner) -induced hamster fibrosarcoma, revealed that the BKV DNA is integrated in the host genome in multiple copies. The sequence of the integrated BKV NCCR was substantially different from (and even contained sequences not found in) that of the BKV (Gardner) strain supposedly used to establish the BKT-1B cell line. PCR amplification, cloning and subsequent sequencing revealed that the original BKV (Gardner) stock contained at least seven different subpopulations of viral genomes. None of them had a control region 'anatomy' which was identical to either the BKV (Gardner) strain, the variant found integrated in BKT-1B cells or any previously published NCCR. In order to study the biological significance of these new BKV NCCR variants we developed a simple cassette model allowing the NCCRs of the new variants to be cloned in an identical genomic background of BKV protein-coding sequences and performed transfection studies with the recombinant genomes in non-permissive rodent cells and in semi-permissive monkey cells. The results demonstrated that the NCCR variants conferred striking differences, in both transforming capacity and host cell permissivity, to the recombinant BKV genomes. Sequence comparisons suggested genetic explanations for these differences, as well as evolutionary relationships between BKV NCCRs.