Human polyomavirus BK (BKV) DNA and proteins have been detected in a number of bone tumours. We therefore investigated whether BKV infection might initiate transformation of human anchorage-dependent osteosarcoma cells in vitro. Infection of the osteosarcoma cell line U-2OS with a naturally occurring BKV strain resulted in soft agarose competent cell clones. In a subclone, designated U-2OS15E, approximately 10-20% of the cells contained episomal BKV genomes. A corresponding proportion of cells expressed BKV proteins and produced viral progeny. This proportion was not increased by BKV superinfection. Furthermore, U-2OS15E cells were resistant to SV40 infection. The transformed status of U-2OS15E cells lasted only for a few passages. However, the persistently infected cells produced infectious virions for more than 300 generations. In addition to representing a model system for persistent BKV infection, the uninfected and persistently BKV-infected cell cultures are useful tools for control and calibration of in situ BKV nucleic acid and protein detection methods.