Cell lines transformed by bovine papillomavirus type 1 DNA were established from transfected primary fibroblast cultures of C57BL/6 mice. Southern blot analysis revealed that most of the viral DNA in the cells was in episomal oligomeric form. These cell lines were tested for tumorigenicity in nude mice in comparison to properties associated with malignant transformation including growth in low-serum medium, focus formation in agar, and resistance to natural killer cells. Two of the 5 cell lines were highly tumorigenic, producing spindle cell sarcomas in all animals. Metastatic spreading to local lymph nodes was also observed. The same cell lines also showed anchorage-independent growth in agar, but no correlation was observed between natural killer cell resistance and tumorigenicity. Viral transcripts were detected in two nontumorigenic and in two tumorigenic cell lines.