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Summary NIH 3T3 mouse fibroblasts were transformed in vitro by transfection with viral DNA from bovine papillomavirus (BPV) types 4, 2 and 1. The viral DNAs were molecularly cloned in pAT153 to construct pBV4, a recombinant plasmid containing the whole genome of BPV4, pBV2 containing the entire genome of BPV2, and pBV1-D1 which contains the 69% transforming DNA fragments of BPV1. The transformed cells lost contact inhibition, were anchorage-independent, required low serum and were tumourigenic in nude mice. This is the first report of cell transformation in vitro by BPV4 DNA. The recombinant plasmids transformed NIH 3T3 cells with an efficiency of 200 to 300 foci/µg DNA. Cleavage of the recombinant plasmids with enzymes that separate the viral DNA from pAT153 DNA did not significantly alter the efficiency of transformation. In all the transformed cells analysed, the recombinant plasmids were found as multiple copies of non-integrated monomers.