The replication of plasmids containing various fragments of the hamster polyomavirus (HaPV) DNA non-coding region was tested in a permissive hamster cell line. We first investigated the importance of some methodological parameters including the time course and the amount of transfecting plasmid DNA and have shown that these factors can greatly influence the relative amount of newly replicated DNA accumulated within the transfected cells. Taking these into account, quantitative comparisons could be made showing the effect of various parts of the regulatory sequence on the HaPV DNA replication.