Transfer of neor and bovine papillomavirus type 1 (BPV1) DNA into rat embryo fibroblasts led to colony formation in G418-containing medium, with no detectable background in controls with neor DNA alone. More than 50% of the drug-resistant clones could be further propagated in culture. The genetic functions of BPV1 involved in colony formation and in long-term immortalization were investigated by both translation termination mutations in the full-length genome, which inactivate individual open reading frames, and constructs in which these open reading frames were separately expressed under control of long terminal repeat promoter enhancers. Expression of either open reading frame E2 or E5 was sufficient for formation of a drug-resistant colony, but long-term growth in culture required that of E6. No significant cooperative effect was observed upon cotransfection of BPV1 and ras oncogene DNAs. Expression of the early region of the human papillomavirus type 16 also led to immortalization of rat embryo fibroblast cells in the same assay, and, unlike what was previously reported in baby rat kidney cells, it required neither activation by a heterologous promoter, nor a cooperating ras oncogene.