The early region of bovine polyomavirus (BPyV) was tested for its cell transformation potential employing an assay of dense focus formation. Dense foci of morphologically transformed cells were observed upon transfection of primary rodent cells with a plasmid construct encoding the complete early region of BPyV under the transcriptional control of the long terminal repeat of Rous sarcoma virus. No transformation of primary rodent cells was observed upon transfection of these cells with a plasmid encoding the complete early region of BPyV under the control of its own transcriptional regulatory sequences. In BPyV-transformed cells, the viral sequences had become integrated into the cellular genome, and expression of large T antigen could be detected in a high percentage of cells. The transformed cells were demonstrated to be capable of anchorage-independent growth and to be oncogenic in immunocompromised newborn rats. Therefore BPyV should be considered as a potentially tumorigenic polyomavirus. Since many commercial batches of calf serum have been shown to be contaminated with BPyV, our observations may have implications for the use of calf serum in cell culture.