The two HindIII fragments of polyoma virus DNA were cloned in the HindIII site of plasmid pBR322, and the biological activity of the recombinant plasmids was tested in tissue culture cells. A mixture of recombinant plasmids containing the HindIII-A and HindIII-B fragments was infectious, but only after cleavage with HindIII. Recombinant plasmids containing the HindIII-A fragment, but not those containing the HindIII-B fragment, induced the transformation of Fischer rat 3T3 cells. These findings indicate that about half of the early region of polyoma virus DNA is not essential for the initiation or the maintenance of transformation.