1. The specific activity of DNA-polymerase alpha isolated from pSV3.neo-transformed cells was more than 9-fold higher than that of polymerase alpha from untransformed cells. 2. Western blot analysis, using anti-SV40 large T antigen, of both a crude cellular extract and of partially purified polymerase alpha from pSV3.neo-transformed cells revealed a single 76 kDa immunoreactive band not found in either crude extracts or partially purified enzyme from untransformed cells. 3. The alpha polymerases from untransformed and transformed cells differed in molecular size, sensitivity to various inhibitors, specificity of template-primer utilization, and binding affinity for DNA cellulose, but showed essentially no differences in Km or Vmax. 4. These data suggest that polymerase alpha isolated from pSV3.neo-transformed cells exhibits altered physical and catalytic characteristics compared with its untransformed cell counterpart, and that those alterations may be associated with increased replication of the genome in plasmid-transformed cells.